WO2022102690A1 - Cell culture system and method of detecting proliferative properties of cells - Google Patents
Cell culture system and method of detecting proliferative properties of cells Download PDFInfo
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- WO2022102690A1 WO2022102690A1 PCT/JP2021/041461 JP2021041461W WO2022102690A1 WO 2022102690 A1 WO2022102690 A1 WO 2022102690A1 JP 2021041461 W JP2021041461 W JP 2021041461W WO 2022102690 A1 WO2022102690 A1 WO 2022102690A1
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
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Definitions
- the present invention relates to a cell culture technique, and particularly to a cell culture system that detects cell proliferation when cells are cultured in a stationary state using a culture vessel made of a gas permeable member.
- the following three methods can be mentioned as methods for confirming the number of cells in the culture vessel.
- a part of the medium in the culture vessel is sampled and the number of cells is measured using a counter or the like.
- the number of cells is measured by image processing the photographs obtained by taking pictures of the cells in the culture vessel with a microscope and a camera.
- the number of cells is measured based on the amount of decrease in dissolved oxygen in the culture vessel.
- the method (1) has a problem that the risk of contamination increases because a part of the culture vessel needs to be opened in order to sample the medium. Further, when culturing adhesive cells, there is a problem that only a part of the cells cannot be exfoliated during the culturing and the number of cells cannot be measured at a desired timing.
- the gas since it is necessary to measure the amount of decrease in dissolved oxygen in the culture vessel, the gas should not move from the inside of the culture vessel to the outside or the gas should not flow into the culture vessel from the outside. It was necessary to keep it sealed. Alternatively, if there is a space on the medium in the culture medium, oxygen dissolution in the medium should be blocked by replacing the space with nitrogen, and the amount of dissolved oxygen consumed by the cells in the medium should be measured. Was needed.
- cell culture is performed using a culture vessel made of a gas permeable member, it is not possible to detect a change in the concentration of only the dissolved oxygen consumed by the cells in the medium, and the inside of the culture vessel is stirred and measured. There was a problem that static culture could not be performed because it was necessary to do so.
- Patent Document 1 discloses a method of measuring the amount of decrease in dissolved oxygen in a sample solution containing a microorganism in a state where the sample solution is sealed in a container. It was necessary to use a culture vessel with no gas permeability. Further, in Patent Document 2, the amount of time fluctuation of the amount of oxygen related to the number of cells in the culture vessel is calculated from the amount of oxygen measured by the oxygen amount measuring means for measuring the amount of oxygen dissolved in the medium in the culture vessel.
- the cell culture apparatus to be used is disclosed. However, this cell culture device uses a closed container in which gas does not flow into the culture container from the outside, and in order to make it measurable from the normal culture state, the space on the medium in the culture container is filled with nitrogen.
- the present invention has been made in view of the above circumstances, and when cells are cultured in a stationary state in a culture vessel made of a gas permeable member, the proliferative potential of the cells in the culture vessel at a desired timing during culture. It is an object of the present invention to provide a cell culture system capable of detecting cells and a method for detecting cell proliferativeness.
- the cell culture system of the present invention is a cell culture system in which cells are cultured in a stationary state by a culture vessel having at least a part of a gas permeable member, and the culture surface in the culture vessel.
- An oxygen concentration sensor that measures the oxygen concentration in the vicinity, the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption per cell. Based on this, the configuration includes a proliferation detection unit that detects the proliferation of the cells.
- the cell culture system of the present invention has a configuration in which the proliferation detection unit calculates the number of cells per unit area as the proliferation of the cells by the following formula (1).
- C G ⁇ (M / 100-D / 100) / S ... Expression (1)
- M Oxygen concentration around the culture vessel (%)
- D Measured value (%) of oxygen concentration sensor
- S Oxygen consumption per unit time per cell (mg / (hr ⁇ ))
- the gradient of the change in the peak value of the increase in oxygen concentration when the growth detection unit added the new medium to the culture vessel a plurality of times and the oxygen concentration when the culture vessel was stabilized It is preferable to use the estimated value of the oxygen concentration in the culture vessel at a desired time point obtained based on the ratio to the slope of the change as the measured value of the oxygen concentration sensor.
- the cell culture system of the present invention has a configuration in which the oxygen concentration around the culture vessel is the oxygen concentration in the incubator in which the culture vessel is housed or the oxygen concentration in the atmosphere.
- the cell culture system of the present invention has a configuration in which the oxygen concentration sensor is a fluorescent type.
- the cell culture system of the present invention has a configuration in which the oxygen concentration sensor is fixed to the culture surface in the culture vessel by using a perforated resin member.
- the method for detecting cell proliferation of the present invention is a method for detecting cell proliferation in a cell culture in which cells are cultured in a stationary state by a culture vessel having at least a part of a gas permeable member.
- the oxygen concentration near the culture surface in the culture vessel is measured by the oxygen concentration sensor arranged on the culture surface in the culture vessel, and the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, and the culture vessel.
- a cell culture system capable of detecting the proliferation of cells in the culture vessel at a desired timing during culture, and a cell culture system. It becomes possible to provide a method for detecting cell proliferation.
- the cell culture system of the present embodiment is a cell culture system in which cells are cultured in a stationary state by a culture vessel having at least a part of a gas permeable member, and measures the oxygen concentration near the culture surface in the culture vessel.
- Proliferation detection that detects cell proliferation based on the oxygen concentration sensor, the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption per cell. It is characterized by having a part.
- the cell culture system of the present embodiment can be configured as shown in FIG. 1, for example.
- the culture container 20 is housed in the incubator 10 and arranged on the mounting table 11.
- the culture container 20 is provided with two ports, and the medium supply container 40 housed in the cold storage 20 is connected to one port by a tube. Further, the waste liquid container 50 in the incubator 10 is connected to the other port of the culture container 20 by a tube.
- Liquid feeding means are arranged on the tube connecting the culture container 20 and the medium supply container 40 and on the tube connecting the culture container 20 and the waste liquid container 50.
- a pump capable of delivering liquid at a low speed and with high accuracy, for example, a perista pump or a syringe pump.
- control device 60 controls the liquid feeding means between the culture vessel 20 and the culture medium supply container 40 at a predetermined timing, and the culture medium is fed from the culture medium supply container 40 to the culture medium 20. Further, the control device 60 controls the liquid feeding means between the culture container 20 and the waste liquid container 50 at a predetermined timing, and the medium is sent from the culture container 20 to the waste liquid container 50.
- Oxygen gas and carbon dioxide gas are supplied to the incubator 10 from the oxygen cylinder O (oxygen supply device) and the carbon dioxide cylinder C (carbon dioxide supply device), respectively, so that the gas concentration inside the incubator 10 is controlled and the culture is performed. It is possible to adjust the gas concentration around the container 20.
- oxygen cylinder O and carbon dioxide cylinder C are connected to the incubator 10 via valves, respectively, and the opening / closing operation of each valve is controlled by the control of the control device 60, and the gas from each gas cylinder to the incubator 10 is controlled.
- the supply of carbon dioxide is controlled so that the oxygen concentration in the incubator 10 can be adjusted.
- the incubator 10 for example, a CO2 incubator or the like can be used.
- the mounting table 11 in the incubator 10 one made of punching metal or the like can be installed and used, and the culture container 20 is arranged on the mounting table 11 and is stored in a closed manner.
- the mounting table 11 may be omitted. Since the cell culture system of the present embodiment can be applied to the case of culturing cells without using the incubator 10, the incubator 10 may be omitted.
- the culture vessel 20 is a closed-type cell culture vessel having gas permeability, which is at least partially composed of a gas-permeable member, and is, for example, by heat-sealing the peripheral portions of two rectangular gas-permeable films.
- the formed one can be used.
- the shape of the culture vessel 20 can be formed so that the shape near the port is inclined with respect to the port, whereby the medium can easily flow to the port. It can also be a rectangle such as a rectangle.
- the number of ports provided in the culture vessel 20 is not limited to two, and may be one or three or more.
- the fluorescent oxygen concentration sensor 21 is arranged on the culture surface in the culture container 20. Further, in the incubator 10, a fluorescence receiving / emitting unit 22 is arranged so that fluorescence can be transmitted / received to / from the fluorescent oxygen concentration sensor 21.
- the fluorescence receiving / emitting unit 22 is arranged, for example, under the mounting table 11, and if the mounting table 11 is transparent, the fluorescent oxygen concentration is provided through the mounting table 11 or through a through hole provided in the mounting table 11. It is possible to send and receive fluorescence to and from the sensor 21.
- the fluorescence receiving / emitting unit 22 may be arranged in contact with the lower side of the culture vessel 20. By controlling the fluorescence receiving / emitting unit 22 by the control device 60, it is possible to measure the oxygen concentration in the culture vessel 20.
- the dissolved oxygen concentration in the medium in the culture vessel 20 varies greatly depending on the location. That is, in the medium near the culture surface (upper surface of the lower gas permeable film 202) where the cells are present, oxygen is actively consumed by the cells, so that oxygen is rapidly consumed and gradually increases as the cell density increases. It becomes 0%. On the other hand, although it depends on the liquid thickness in the culture container 20, for example, when the liquid thickness is 10 mm or more, the upper surface in the culture container 20 on the opposite side of the culture surface (lower surface of the upper surface gas permeable film 201). In the nearby medium, the oxygen concentration does not decrease easily from the initial state (for example, about 21% in the atmosphere) when the cells are not present in the vicinity. In the medium near the center of the culture vessel 20, the oxygen concentration is in the middle of these (for example, 10%).
- the oxygen concentration in the culture vessel 20 based on the oxygen consumption by the cells is appropriately measured. Is possible. Further, as will be described later, in the cell culture system of the present embodiment, the oxygen consumption by the cells is appropriately calculated by adding the oxygen permeability of the culture vessel 20 and the oxygen concentration around the culture vessel 20. It makes it possible. Further, according to the cell culture system of the present embodiment, even when the oxygen concentration in the culture vessel 20 continues to be about 0% to 1% and it is difficult to measure the oxygen concentration by the fluorescent oxygen concentration sensor 21. It makes it possible to properly estimate the amount of oxygen consumed by cells.
- the dissolved gas in the medium in the culture vessel is dissolved at a concentration that is in equilibrium with the partial pressure around the culture vessel.
- the concentration of oxygen dissolved in the medium placed in the incubator is about 21%, which is the same as the concentration in the atmosphere when the gas concentration in the incubator is not controlled.
- the gas permeability (velocity) of a film is determined by the difference in gas partial pressure between both sides of the film, the affinity between the film and the gas, and the diffusivity of moving gas molecules into the film.
- the fluorescent substance absorbs the light and becomes an excited state, and returns to the ground state while radiating fluorescence.
- the excitation energy is deprived and the fluorescence emission intensity is lowered. Therefore, the more oxygen there is, the smaller the fluorescence emission intensity.
- the fluorescence receiving / emitting unit measures the amount of oxygen based on the change in emission intensity depending on the number of oxygen molecules in contact with the oxygen molecule in contact with the oxygen dissolved in the medium.
- a resin film or the like can be preferably used, and a polyolefin-based resin such as polyethylene or polypropylene can be used.
- a polyolefin-based resin such as polyethylene or polypropylene
- polyethylene, a copolymer of ethylene and ⁇ -olefin, a copolymer of ethylene and vinyl acetate, an ionomer using ethylene and acrylic acid, a methacrylic acid copolymer and metal ions, and the like can be mentioned.
- polyolefins, styrene-based elastomers, polyester-based thermoplastic elastomers and the like can also be used.
- soft vinyl chloride resin polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomer, polyester-based thermoplastic elastomer, silicone-based thermoplastic elastomer, styrene-based elastomer, for example, SBS (styrene).
- SBS styrene
- SIS styrene / isoprene / styrene
- SEBS styrene / ethylene / butylene / styrene
- SEPS styrene / ethylene / propylene / styrene
- polyolefin resin fluororesin and the like
- the gas permeable member used for at least a part of the culture vessel 20 it is preferable to use a thermoplastic resin having particularly excellent gas permeable performance among the above materials, for example, a polyolefin such as polyethylene such as LLDPE or polypropylene. A based resin can be preferably used. Further, the gas permeable member is preferably a transparent material.
- the fluorescent oxygen concentration sensor 21a is used as a culture surface in the culture container 20a (in the example of the figure, the upper surface of the lower surface gas permeable film 202a). ) Is preferably fixed using a perforated resin member 23a. At this time, the fluorescent oxygen concentration sensor 21a is sandwiched between the perforated resin member 23a and the culture surface, and the welding region H of the perforated resin member 23a can be welded and fixed to the culture surface.
- a polyolefin resin such as polyethylene such as LLDPE or polypropylene
- polyethylene a copolymer of ethylene and ⁇ -olefin, a copolymer of ethylene and vinyl acetate, an ionomer using ethylene and acrylic acid, a methacrylic acid copolymer and metal ions, and the like can be mentioned.
- the perforated resin member 23a is for contacting the fluorescent oxygen concentration sensor 21a with the medium while fixing the fluorescent oxygen concentration sensor 21a to the culture surface, a mesh-like material can be preferably used. Further, as the perforated resin member 23a, a porous material such as a sponge can also be used.
- the specific mesh-like material and porous material are not particularly limited, but those having a melting point close to that of the culture vessel 20a and the same material are preferable, for example, a polyethylene mesh material (NBC Mesh Tech Co., Ltd., PE200). (Eyes, etc.) and polyethylene porous (Teijin Co., Ltd., Milime, large diameter ⁇ 3 ⁇ m) can be preferably used.
- the medium can be injected into the culture vessel 20a or from the culture vessel 20a. It is possible to prevent the fluorescent oxygen concentration sensor 21a from coming off and moving when the medium is discharged.
- the cells to be cultured using the culture vessel 20 are not particularly limited, and even floating cells such as lymphocytes and dendritic cells that are suspended in the culture medium and cultured can be placed on the culture surface in the culture vessel.
- Artificial pluripotent stem cells iPS cells
- neural stem cells iPS cells
- embryonic stem cells ES cells
- mesenchymal stem cells hepatocytes
- pancreatic islet cells myocardial cells
- corneal endothelial cells a cell that are adhered and cultured.
- activation steps that are adhered and cultured. It may be an adhesive cell such as a lymphocyte.
- the cells to be cultured using the culture vessel 20 may be those in which aggregates such as spheroids and organoids are formed, and the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells are aggregated. It can also be applied to mass culture.
- control device 60 in the cell culture system of the present embodiment will be described in detail.
- the control device 60 (control unit) has an input / output unit 61, a fluorescent sensor input / output unit 62, a control unit 63, an operation unit 64, and a power supply unit 65.
- the input / output unit 61 is connected to a pump arranged between the culture container 20 and the medium supply container 40 and a pump arranged between the culture container 20 and the waste liquid container 50, and is based on the input information from the control unit 63. And control the operation of these pumps. As a result, the medium is supplied to the culture container 20 from the medium supply container 40, and the medium is discharged from the culture container 20 to the waste liquid container 50.
- the input / output unit 61 is connected to the gas sensor 12 of the incubator 10, the valve arranged between the oxygen cylinder O and the incubator 10, and the valve arranged between the carbon dioxide cylinder C and the incubator 10, and the gas sensor 12 is connected.
- the input information from is transmitted to the control unit 63.
- the input / output unit 61 controls the operation of these valves based on the input information from the control unit 63.
- oxygen gas can be supplied into the incubator 10 from the oxygen cylinder O
- carbon dioxide gas can be supplied into the incubator 10 from the carbon dioxide cylinder C
- the gas concentration in the incubator 10 can be controlled to a desired level. Is possible.
- the fluorescence sensor input / output unit 62 is connected to the fluorescence light receiving / emitting unit 22, controls the emission of fluorescence by the fluorescence receiving / emitting unit 22 based on the input information from the control unit 63, and receives light from the fluorescent oxygen concentration sensor 21. Information based on this is transmitted to the control unit 63. As a result, the oxygen concentration in the vicinity of the culture surface in the culture vessel 20 can be measured, and the control unit 63 can use this oxygen concentration for detecting the proliferativeness of the cells.
- the control unit 63 is configured by a PLC (programmable logic controller) or the like, and can pre-program and store desired control contents, and can control the operation of each unit based on this. That is, the control unit 63 transmits information for controlling the pump or valve to the input / output unit 61 at a predetermined timing. Further, it is also possible to transmit information for controlling the pump or valve to the input / output unit 61 based on the input information from the fluorescent sensor input / output unit 62, the time information, and various other information.
- PLC programmable logic controller
- control unit 63 includes a growth detection unit 631 for detecting the proliferation of cells in the culture vessel 20.
- the proliferation detection unit 631 detects the proliferation of cells based on the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption per cell. do. Specifically, the proliferation detection unit 631 can calculate the number of cells per unit area as the proliferation of cells.
- the oxygen permeability of the gas permeable member and the oxygen consumption per cell are calculated in advance according to the intended use and stored in the growth detection unit 631 or the like. As will be described later, the oxygen consumption of one PBMC cell was calculated to be 1.0e-9 mg / (hr ⁇ ). In addition, as a result of calculating the oxygen consumption of one Jurkat (lymphocyte-strained cell) cell, it was 1.74e-9 mg / (hr ⁇ cell).
- the actual culture form Prior to determining the oxygen permeability (mg / (cm 2 ⁇ hr ⁇ atm)), the actual culture form is first determined. That is, the oxygen permeability can be calculated as follows after determining the container size, the amount of the filling medium, the position where the fluorescent sensor is installed, and whether or not the pressing jig is used.
- the culture container 20 As the culture container 20, the culture bag in Test 1 described later having a bottom area of 50 cm 2 in which two polyethylene films having a thickness of 110 ⁇ m are bonded is used, and the fluorescent oxygen concentration sensor 21 is fixed to substantially the center of the inner surface of the culture bag. Then, it is assumed that the culture vessel 20 is sandwiched between the pressing jigs and the culture is performed in a pressurized state.
- the bottom surface of the pressing jig is a transparent resin plate, and the fluorescence receiving / emitting unit 22 is fixed at a position corresponding to the fluorescent oxygen concentration sensor 21 on the resin plate.
- the medium is then heated to the culture temperature of 37 ° C. and injected into the culture vessel 20 together with the cells.
- PBMC Peripheral Blood Mononuclear Cells
- Jurkat Lymphocytic cells
- Oxygen permeability was calculated and the average value was obtained.
- As the medium for PBMC AlyS505N-7 (manufactured by Cell Science Laboratory Co., Ltd.) to which 2% of FBS was added was used, and 35 ml of the medium was filled in the culture bag together with the cells.
- As the Jurkat medium AlyS505N-0 (manufactured by Cell Science Laboratory Co., Ltd.) to which FBS 2% was added was used, and 35 ml of the medium was filled in the culture bag together with the cells.
- the cell filling amount per unit area of PBMC was 2.1 e6 cells / cm 2 , and the cells sank approximately 2 hours after filling the culture bag, and the oxygen concentration became stable. The oxygen concentration at that time was 6%.
- the oxygen permeability G can be calculated as follows.
- G Cell filling amount per unit area x
- the oxygen concentration around the culture bag was controlled to 30%. After about 2 hours, the cells sank and the oxygen concentration became stable. The oxygen concentration at that time was 2%.
- the oxygen permeability G can be calculated as follows.
- G Cell filling amount per unit area x
- the oxygen permeability G can be calculated as follows.
- G Cell filling amount per unit area x
- the cell filling amount per unit area of Jurkat was 1.9 e6 cells / cm 2 , and after filling the culture bag, the oxygen concentration around the culture bag was controlled to 30%. After about 2 hours, the cells sank and the oxygen concentration became stable. The oxygen concentration at that time was 5%.
- the oxygen permeability G can be calculated as follows.
- G Cell filling amount per unit area x
- the oxygen permeability G of the culture bag used under the above conditions was determined as follows.
- G (1.40e-2 + 1.43e-2 + 1.47e-2 + 1.32e-2)
- / 4 1.4e-2 (mg / (cm 2 ⁇ hr ⁇ atm))
- the oxygen consumption per cell (mg / (hr ⁇ )) can be calculated as follows. First, cells and a medium are injected into a closed container that does not permeate gas with the number of cells grasped in advance. Use a syringe or the like to discharge air bubbles so that air bubbles are not mixed in the closed container as much as possible. Calculate the amount of dissolved oxygen from the volume of the closed container.
- a closed container having a volume of 10 ml was filled with a medium heated to 37 ° C. 6.86e-2mg of oxygen is dissolved in 10ml of medium at 37 ° C (atmospheric oxygen concentration of about 21%) (6.86e-2mg / l @ 37 ° C (saturated dissolved oxygen)).
- the change in oxygen concentration in the closed container during culture is measured, and the decrease concentration of dissolved oxygen per hour is calculated.
- PBMC is injected into a 10 ml closed container with a cell filling amount of 2e6 cells / ml per unit area, 2e7 cells float in the container.
- the decrease in oxygen concentration at that time was 0.1% / min, and decreased by 6% per hour.
- the measured value of the oxygen concentration sensor is the oxygen concentration near the culture surface in the culture vessel 20 measured by the fluorescent oxygen concentration sensor 21, and the growth is detected from the fluorescent light receiving / receiving unit 22 via the fluorescent sensor input / output unit 62. It is input to the unit 631.
- the oxygen concentration around the culture container is the oxygen concentration in the incubator 10 in which the culture container 20 is housed.
- the oxygen concentration in the incubator 10 is not controlled, or when the incubator 10 is not used, it is the oxygen concentration in the atmosphere (about 21%). This is preset in the control unit 63.
- G ⁇ (M / 100-D / 100) C ⁇ S
- G Oxygen permeability of gas permeable member (mg / (cm 2 ⁇ hr ⁇ atm))
- M Oxygen concentration around the culture vessel (%)
- D Measured value (%) of oxygen concentration sensor
- C Number of cells per unit area (pieces / cm 2 )
- S Oxygen consumption per unit time per cell (mg / (hr ⁇ ))
- the growth detection unit 631 can calculate the number of cells per unit area as the proliferative property of the cells by the following formula (1).
- C G ⁇ (M / 100-D / 100) / S ... Expression (1)
- D Measured value (%) of oxygen concentration sensor
- S Oxygen consumption per unit time per cell (mg / (hr ⁇ ))
- the number of cells per unit area calculated by the cell culture system of the present embodiment does not strictly measure the actual number of cells, but estimates the number of cells roughly based on the culture environment of the culture vessel 20. It is a thing. In mass culture of cells, it is important to confirm that the culture is performed properly, so it is very useful to estimate the number of cells in the culture vessel.
- the growth detection unit 631 shows the gradient of the change in the peak value of the increase in oxygen concentration when the new medium is added to the culture vessel 20 multiple times, and the oxygen when the culture vessel 20 is stable. It is also preferable to use the estimated value of the oxygen concentration in the culture vessel 20 at a desired time point obtained based on the ratio with the gradient of the change in concentration as the measured value D of the oxygen concentration sensor of the above formula (1). ..
- the culture at a desired time point obtained based on the ratio of the slope of the change in the peak value of the increase in oxygen concentration when the new medium was added to the culture bag multiple times and the slope of the change in the oxygen concentration when the culture bag was stable.
- D'of the oxygen concentration in the bag the estimated value of the oxygen concentration sensor
- the rise in oxygen concentration when a new medium is added The oxygen concentration at the peak is affected by the temperature of the medium to be added. Therefore, it is preferable to keep the temperature of the medium constant and add the medium. Further, the calculation method of the estimated value D'will be described in detail in Test 5.
- the operation unit 64 includes a display unit such as a touch panel, transmits input information by the user to the control unit 63, and executes PLC setting and the like. In addition, the input information from the control unit 63 is displayed.
- the power supply unit 65 (stabilized power supply or the like) supplies electricity to each unit in the control device 60.
- the control device 60 may be further provided with a relay and a molded case circuit breaker. Further, a part or all of the configuration of the control unit 63, the operation unit 64, etc. in the control device 60 may be realized by a microcomputer or a computer.
- the cell proliferative detection method of the present embodiment is a cell proliferative detection method in cell culture in which cells are cultured in a stationary state by a culture vessel having at least a part of a gas permeable member, and is a culture vessel.
- the oxygen concentration near the culture surface in the culture vessel is measured by the oxygen concentration sensor placed on the culture surface inside, and the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, It is characterized by calculating the number of cells per unit area based on the amount of oxygen consumed per cell.
- the number of cells per unit area can be calculated by the following formula (1).
- C G ⁇ (M / 100-D / 100) / S ... Expression (1)
- S Oxygen consumption per unit time per cell (mg / (hr ⁇ ))
- the cell culture system of the present embodiment and the method for detecting cell proliferation when cells are cultured in a static state in a culture vessel made of a gas permeable member, the desired timing during the culture is achieved.
- the proliferation of cells in the culture vessel can be detected, and the number of cells per unit area can be calculated as the proliferation of cells. Therefore, according to the present embodiment, it is possible to confirm whether or not the culture is properly performed in the mass culture of cells.
- Test 1 Jurkat (lymphocyte cell line) was cultured to detect proliferativeness. In this test, the cells were continuously cultured without interruption during the test period, and the estimated value of the cell density was compared with the calculated value of the number of cells per unit area according to the present embodiment.
- Test 2 PBMC (Peripheral Blood Mononuclear Cells) was cultured, and the measured value of the cell density after the culture was compared with the calculated value of the number of cells per unit area according to the present embodiment. rice field.
- Test 3 PBMC was cultured, the culture was interrupted at a predetermined timing, the actual number of cells was counted, the measured value of the obtained cell density and the calculated value of the number of cells per unit area according to the present embodiment. was compared.
- Test 4 iPS cells were cultured, the actual number of cells was counted after the culture was completed, and the measured value of the obtained cell density was compared with the calculated value of the number of cells per unit area according to the present embodiment. ..
- Test 5 is for the continuation period of the culture by the test 3, and includes the period in which the cell density in the culture vessel becomes high and the measured value of the oxygen concentration near the culture surface is less than 1%. After the culture was completed, the actual number of cells was counted, and the measured value of the obtained cell density was compared with the calculated value of the number of cells per unit area according to the present embodiment.
- a culture bag As a culture bag, a culture bag (manufactured by Toyo Seikan Group Holdings Co., Ltd.) made of linear low-density polyethylene having a thickness of 110 ⁇ m, having an outer size of 120 mm ⁇ 65 mm and a bottom area of the culture space of 48 cm 2 was prepared. The culture bag was formed by heat-sealing the edges of two flat films. The oxygen permeability in this culture bag was 1.4e-2 (mg / (cm 2 ⁇ hr ⁇ atm)).
- a fluorescent oxygen concentration sensor (sensor chip PSt3 type, PreSens) was fixed in the center of the inner surface of the culture bag. This culture bag was sandwiched between pressing jigs and cultured under pressure. The bottom surface of the pressing jig is a transparent resin plate, and the fluorescence receiving / emitting unit is fixed at a position corresponding to the fluorescent oxygen concentration sensor on the resin plate. The fluorescence receiving / emitting unit was attached to the fluorescence sensor input / output unit in the control device, and OXY-4mini (Titec Co., Ltd.) was used as the fluorescence sensor input / output unit. The same applies to the fluorescent oxygen concentration sensor, the fluorescent light receiving / emitting unit, and the fluorescent sensor input / output unit used in the following tests.
- Jurkat cell line derived from human leukemia T cells
- AlyS505N-0 manufactured by Cell Science Laboratory Co., Ltd.
- FBS 2% was added
- a multi-gas incubator capable of high oxygen culture (PHC Corporation, model MCO-5M-PJ) was used, and an automatic liquid feeding mechanism was installed in the incubator.
- the medium supply bag filled with the medium was stored in a cold storage at 10 ° C. and connected to the culture bag with a tube.
- the waste liquid bag was connected to the culture bag with a tube.
- the medium was fed under the control of a tube pump.
- the medium was warmed while passing through the 37 ° C incubator to about 30 ° C by the time it entered the culture bag.
- the control of the tube pump and the control of the oxygen concentration of the incubator were performed by the control unit in the control device.
- the proliferation detection unit in the control unit was configured by a computer.
- the medium deteriorated by the culture was discharged from the medium bag and filled with a new medium. Specifically, the medium was exchanged at a predetermined timing from the start of the culture. At this time, the amount of medium discharged was 25 ml out of 35 ml. The liquid feeding rate was 2 ml / min. When new medium was added by medium exchange, the oxygen concentration in the culture bag temporarily increased, and a peak was displayed in the graph described later.
- the oxygen concentration in the incubator was increased at the specified timing. Specifically, it was changed from 21% to 25% at the timing 23 hours after the start of culture. In addition, it was changed from 25% to 30% at the timing 32 hours after the start of culture. In addition, it was changed from 30% to 35% at the timing 50 hours after the start of culture. In addition, it was changed from 35% to 40% at the timing 53 hours after the start of culture.
- FIG. 4 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of Test 1. Based on this measurement result, the estimated value of the cell density and the calculated value of the number of cells per unit area according to this embodiment were compared with each other 10, 40, and 60 hours after the start of the culture.
- the calculated values of the number of cells per unit area by the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells are calculated as follows after 10, 40, and 60 hours, respectively.
- the oxygen permeability (mg / (cm 2 ⁇ hr ⁇ atm)) of the gas permeable member was 1.4e-2.
- the oxygen consumption per unit time (mg / (hr ⁇ )) of one cell was 1.74e-9.
- the estimated value of the cell density after 40 hours was 2.13e6 cells / cm 2 as described above.
- the estimated value of the cell density after 60 hours was 2.82e6 cells / cm 2 as described above.
- the desired timing during culture is achieved. It was found that the approximate number of cells per unit area in the cell can be calculated.
- Test 2 The same culture bag as in Test 1 was prepared.
- the oxygen permeability in this culture bag was 1.4e-2 (mg / (cm 2 ⁇ hr ⁇ atm)).
- a fluorescent oxygen concentration sensor was fixed in the center of the inner surface of the culture bag.
- This culture bag was sandwiched between pressing jigs and cultured under pressure.
- the bottom surface of the pressing jig is a transparent resin plate, and the fluorescence receiving / emitting unit is fixed at a position corresponding to the fluorescent oxygen concentration sensor on the resin plate.
- PBMC Peripheral Blood Mononuclear Cells
- AlyS505N-7 manufactured by Cell Science Laboratory Co., Ltd.
- FBS 2% FBS 2% was added
- 35 ml of the medium was filled in the culture bag together with the cells.
- the oxygen concentration was not measured for 3 days after sowing in the culture bag, and the oxygen concentration in the culture bag was measured for about 60 hours for the following 3 days.
- a multi-gas incubator capable of high oxygen culture (PHC Corporation, model MCO-5M-PJ) was used, and an automatic liquid feeding mechanism was installed in the incubator.
- the medium supply bag filled with the medium was stored in a cold storage at 10 ° C. and connected to the culture bag with a tube.
- the waste liquid bag was connected to the culture bag with a tube. The medium was fed under the control of a tube pump.
- the medium deteriorated by the culture was discharged from the medium bag and filled with a new medium. Specifically, the medium was exchanged at a predetermined timing from the start of the culture. At this time, the amount of medium discharged was 25 ml out of 35 ml. The liquid feeding rate was 2 ml / min.
- the oxygen concentration in the culture bag temporarily increased, and a peak was displayed in the graph described later.
- the oxygen concentration of the incubator 10 was not increased, and the culture in high oxygen was not performed.
- FIG. 5 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of Test 2.
- the calculated value of the number of cells per unit area by the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells is calculated as follows after 61 hours.
- the oxygen permeability (mg / (cm 2 ⁇ hr ⁇ atm)) of the gas permeable member was 1.4e-2.
- the oxygen consumption per unit time (mg / (hr ⁇ )) of one cell was 1.00e-9.
- the cell culture system of the present embodiment and the method for detecting cell proliferation, when cells are cultured in a stationary state in a culture vessel made of a gas permeable member, the cell unit. It was found that an approximate number of cells per area could be calculated.
- Test 3 The same culture bag as in Test 1 was prepared.
- the oxygen permeability in this culture bag was 1.4e-2 (mg / (cm 2 ⁇ hr ⁇ atm)).
- a fluorescent oxygen concentration sensor was fixed in the center of the inner surface of the culture bag.
- This culture bag was sandwiched between pressing jigs and cultured under pressure.
- the bottom surface of the pressing jig is a transparent resin plate, and the fluorescence receiving / emitting unit is fixed at a position corresponding to the fluorescent oxygen concentration sensor on the resin plate.
- PBMC Peripheral Blood Mononuclear Cells
- PBMCs Prior to seeding in culture bags, PBMCs were activated for 3 days using a flask coated with anti-CD3 antibody and solidified, and then seeded in culture bags at a cell density of 1.06e6 cells / cm 2 .
- AlyS505N-7 manufactured by Cell Science Laboratory Co., Ltd.
- FBS 2% FBS 2% was added
- 35 ml of the medium was filled in the culture bag together with the cells.
- the oxygen concentration was not measured for 4 days after sowing in the culture bag, and the oxygen concentration in the culture bag was measured for about 65 hours for the following 3 days.
- Test 3 unlike Test 2, in order to measure the number of cells, the automatic liquid feeding was stopped, the culture bag was stirred to uniformly suspend the cells in the medium, and the medium was removed from the culture bag using a syringe. Approximately 0.1 ml was collected, stained with tripan blue, and the cell density was measured using a hemocytometer. The number of collected cells is extremely small, and there is no effect on the overall cell density. The use of the incubator and the exchange of the medium were carried out in the same manner as in Test 1.
- FIGS. 6 and 7 show graphs showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of Test 3.
- the graph of FIG. 6 shows the measurement result of the oxygen concentration up to the first measurement of the cell number
- the graph of FIG. 7 shows the measurement result of the oxygen concentration up to the second measurement of the cell number.
- the oxygen concentration of the incubator 10 was increased at a specified timing. Specifically, in the graph of FIG. 6, it was changed from 21% to 30% at the timing 4 hours after the start of measurement. In addition, it was changed from 30% to 40% at the timing 23 hours after the start of measurement. Furthermore, in the graph of FIG. 7, it was changed from 40% to 50% at the timing 6 hours after the start of measurement.
- the culture bag was stirred to uniformly float the cells in the medium, about 0.1 ml of the medium was collected from the culture bag, and stained with tripan blue to obtain a hemocytometer. The cell density was measured using. As a result, the cell density 42 hours after the start of measurement was 4.04e6 cells / cm 2 .
- the culture bag was stirred to uniformly float the cells in the medium, about 0.1 ml of the medium was collected from the culture bag, stained with tripan blue, and a hemocytometer was obtained. The cell density was measured using. As a result, the cell density 22.5 hours after the start of measurement was 5.2e6 cells / cm 2 .
- the calculated value of the number of cells per unit area by the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells is calculated as follows. First, the oxygen permeability (mg / (cm 2 ⁇ hr ⁇ atm)) of the gas permeable member is 1.4e-2. The oxygen consumption per unit time (mg / (hr ⁇ )) of one cell was 1.00e-9.
- the cell culture system of the present embodiment and the method for detecting cell proliferation, when cells are cultured in a stationary state in a culture vessel made of a gas permeable member, the cell unit. It was found that an approximate number of cells per area could be calculated.
- a culture bag As a culture bag, a culture bag (manufactured by Toyo Seikan Group Holdings Co., Ltd.) made of linear low-density polyethylene having a thickness of 110 ⁇ m, having an outer size of 120 mm ⁇ 65 mm and a bottom area of the culture space of 48 cm 2 was prepared.
- this culture bag is provided with a plurality of protrusions on the inside and a plurality of small protrusions are formed on the outer surface.
- the peripheral edges of two films processed in parallel in a mountain range with a pitch of 0.11 mm, a height of 0.19 mm, and an angle of 75 ° are heat-sealed. Formed by The thickness of the thinnest part (thin wall part) of the film is 25 ⁇ m.
- the oxygen permeability in this culture bag was 8.0e-2 (mg / (cm 2 ⁇ hr ⁇ atm)). The oxygen permeability of this culture bag was calculated in the same manner as described above using the culture bag used in Test 1 as an example.
- a fluorescent oxygen concentration sensor was fixed in the center of the inner surface of the culture bag. Moreover, in this test, the culture was carried out without using a pressing jig. A fluorescence receiving / emitting unit was placed at a position corresponding to the fluorescent oxygen concentration sensor on the lower side of the culture bag.
- iPS cells 1231A3 strain, Center for iPS Cell Research and Application, Kyoto University
- iPS cells 1231A3 strain, Center for iPS Cell Research and Application, Kyoto University
- both the upper surface and the lower surface in the culture bag are used as the culture surface, and the seeded cell density is for both sides.
- StemFit (R) AK-02N, Ajinomoto Healthy Supply Co., Ltd.
- 35 ml of the medium was filled in the culture bag together with the cells.
- a multi-gas incubator capable of high oxygen culture (PHC Corporation, model MCO-5M-PJ) was used.
- the medium was changed manually without using the automatic liquid feeding mechanism.
- the medium supply bag filled with the medium was stored in a cold storage at 10 ° C. and connected to the culture bag with a tube.
- the waste liquid bag was connected to the culture bag with a tube.
- the medium was fed under the control of a tube pump. The medium was warmed while passing through the 37 ° C incubator to about 30 ° C by the time it entered the culture bag.
- the medium deteriorated by the culture was discharged from the medium bag and filled with a new medium. Specifically, the medium was exchanged at a predetermined timing from the start of the culture. At this time, the amount of medium discharged was 35 ml out of 35 ml, and the liquid feeding rate was 2 ml / min.
- the oxygen concentration in the culture bag temporarily increased, and a peak was displayed in the graph described later.
- the oxygen concentration of the incubator 10 was not increased, and the culture in high oxygen was not performed.
- FIG. 9 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of Test 4.
- the calculated value of the number of cells per unit area by the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells is calculated as follows. First, the oxygen permeability (mg / (cm 2 ⁇ hr ⁇ atm)) of the gas permeable member was 8.0e-2. The oxygen consumption per unit time (mg / (hr ⁇ )) of one cell was 2.34e-9.
- the cell culture system of the present embodiment and the method for detecting cell proliferation, when cells are cultured in a stationary state in a culture vessel made of a gas permeable member, the cell unit. It was found that an approximate number of cells per area could be calculated.
- Test 5 This test was carried out in succession to Test 3, and the oxygen concentration in the culture bag was measured for about 60 hours for 3 days following the measurement of the oxygen concentration in Test 3.
- FIG. 10 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of Test 5.
- the culture bag was stirred to uniformly float the cells in the medium, about 0.1 ml of the medium was collected from the culture bag, stained with trypan blue, and the cell density was measured using a blood cell counter. As a result, the cell density 56 hours after the start of measurement was 1.04e7 cells / cm 2 .
- the cell density in the culture bag is extremely high after 25 hours from the start of measurement.
- the oxygen concentration in the culture bag was less than 1%.
- the number of cells per unit area is calculated by the following method. First, in the culture time before the cell density in the culture bag becomes very high, the gradient PT of the change in the peak value of the increase in oxygen concentration when the new medium is added to the culture bag multiple times, and the stability of the culture bag. The gradient QT of the change in oxygen concentration at time is calculated, and the ratio (QT / PT) is calculated.
- the ratio (QT / PT) is 2.9.
- the peak immediately before Q4 (less than 56, 1) 56 hours after the start of measurement is P4 (55.1, 22.3), and the peak immediately before Q3 (31.1, 2.3) when the oxygen concentration can be measured is P3 (55.1, 22.3). 29.5, 32.7).
- the culture at a desired time point obtained based on the ratio of the slope of the change in the peak value of the increase in oxygen concentration when the new medium was added to the culture bag multiple times and the slope of the change in the oxygen concentration when the culture bag was stable.
- D'of the oxygen concentration in the bag the estimated value of the oxygen concentration sensor
- the number of cells per unit area is calculated as follows.
- the oxygen permeability (mg / (cm 2 ⁇ hr ⁇ atm)) of the gas permeable member was 1.4e-2.
- the oxygen consumption per unit time (mg / (hr ⁇ )) of one cell was 1.00e-9.
- the cell culture system of the present embodiment and the method for detecting cell proliferation, when cells are cultured in a stationary state in a culture vessel made of a gas permeable member, the cell unit. It was found that an approximate number of cells per area could be calculated.
- the culture container is not limited to the one illustrated, and for example, a container having one port, a container having three ports, a container having a plurality of wells on the culture surface, and the like, which are appropriately modified according to the purpose of the culture, are used. It is possible.
- the present invention can be suitably used when cells are mass-cultured at high density using a cell culture bag.
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Abstract
Provided is a cell culture system by which, when cells are statically cultured in a culture container made of a gas permeable material, the proliferative properties of the cells in the culture container can be detected at a desired timing during the culture. This cell culture system, which is for statically culturing cells in a culture container 20 that is at least partly made of a gas permeable material, comprises an oxygen concentration sensor 21 that measures the oxygen concentration in the vicinity of the culture surface in the culture container 20, and a proliferation detection section 631 that detects the proliferative properties of the cells on the basis of the oxygen permeability of the gas permeable material, the value measured by the oxygen concentration sensor, the oxygen concentration around the culture container 20 and the oxygen consumption per cell.
Description
本発明は、細胞培養技術に関し、特にガス透過性部材からなる培養容器によって細胞を静置状態で培養する場合における細胞の増殖性を検出する細胞培養システムに関する。
The present invention relates to a cell culture technique, and particularly to a cell culture system that detects cell proliferation when cells are cultured in a stationary state using a culture vessel made of a gas permeable member.
近年、医薬品の生産や、遺伝子治療、再生医療、免疫療法等の分野において、細胞や組織などを人工的な環境下で効率良く大量に培養することが求められている。
このような状況において、ガス透過性部材からなる袋状の培養容器を用いて閉鎖系で細胞を自動的に大量培養することが提案されている。 In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, etc., it has been required to efficiently and mass-culture cells and tissues in an artificial environment.
Under such circumstances, it has been proposed to automatically mass-culture cells in a closed system using a bag-shaped culture vessel made of a gas-permeable member.
このような状況において、ガス透過性部材からなる袋状の培養容器を用いて閉鎖系で細胞を自動的に大量培養することが提案されている。 In recent years, in the fields of pharmaceutical production, gene therapy, regenerative medicine, immunotherapy, etc., it has been required to efficiently and mass-culture cells and tissues in an artificial environment.
Under such circumstances, it has been proposed to automatically mass-culture cells in a closed system using a bag-shaped culture vessel made of a gas-permeable member.
細胞の大量培養にあたっては、細胞の増殖性を検出して、培養が適切に行われているかを確認することが重要である。また、その細胞の増殖性の検出のために、例えば培養中の所望のタイミングにおける培養容器内の細胞数を確認可能にすることが望ましい。
In mass culturing of cells, it is important to detect the proliferativeness of the cells and confirm that the culturing is performed properly. Further, in order to detect the proliferativeness of the cells, it is desirable to be able to confirm, for example, the number of cells in the culture vessel at a desired timing during culture.
培養容器内の細胞数を確認する手法として、以下の3つを挙げることができる。
(1)培養容器内の培地の一部を採取(サンプリング)して、計数盤等を用いて細胞数を計測する。
(2)培養容器内の細胞を顕微鏡とカメラにより撮影して得られた写真を画像処理することで細胞数を計測する。
(3)培養容器内における溶存酸素の減少量にもとづき細胞数を計測する。 The following three methods can be mentioned as methods for confirming the number of cells in the culture vessel.
(1) A part of the medium in the culture vessel is sampled and the number of cells is measured using a counter or the like.
(2) The number of cells is measured by image processing the photographs obtained by taking pictures of the cells in the culture vessel with a microscope and a camera.
(3) The number of cells is measured based on the amount of decrease in dissolved oxygen in the culture vessel.
(1)培養容器内の培地の一部を採取(サンプリング)して、計数盤等を用いて細胞数を計測する。
(2)培養容器内の細胞を顕微鏡とカメラにより撮影して得られた写真を画像処理することで細胞数を計測する。
(3)培養容器内における溶存酸素の減少量にもとづき細胞数を計測する。 The following three methods can be mentioned as methods for confirming the number of cells in the culture vessel.
(1) A part of the medium in the culture vessel is sampled and the number of cells is measured using a counter or the like.
(2) The number of cells is measured by image processing the photographs obtained by taking pictures of the cells in the culture vessel with a microscope and a camera.
(3) The number of cells is measured based on the amount of decrease in dissolved oxygen in the culture vessel.
しかしながら、(1)の手法には、培地をサンプリングするために培養容器の一部を開放する必要があるため、コンタミネーションの危険性が増大するという問題があった。また、接着系細胞を培養する場合、培養の途中で細胞の一部のみを剥離することができず、所望のタイミングで細胞数を計測できないという問題があった。
However, the method (1) has a problem that the risk of contamination increases because a part of the culture vessel needs to be opened in order to sample the medium. Further, when culturing adhesive cells, there is a problem that only a part of the cells cannot be exfoliated during the culturing and the number of cells cannot be measured at a desired timing.
また、(2)の手法には、浮遊系細胞を培養する場合、培養密度が小さいときは計測可能であるが、培養の中盤以降において細胞密度が高くなると細胞が積層してしまうため、計測することが困難になるという問題があった。また、この手法では顕微鏡とカメラと画像処理装置が必要になるため、細胞培養システムの機構が複雑になってしまうという問題もあった。
In addition, in the method (2), when culturing floating cells, it is possible to measure when the culture density is low, but when the cell density becomes high after the middle of the culture, the cells are stacked, so measurement is performed. There was the problem that it became difficult. In addition, this method requires a microscope, a camera, and an image processing device, which causes a problem that the mechanism of the cell culture system becomes complicated.
さらに、(3)の手法は、培養容器内における溶存酸素の減少量を測定する必要があるため、培養容器内からガスが外部に移動したり、外部から培養容器内にガスが流入しないように密閉状態にすることが必要であった。あるいは、培養容器内の培地上に空間が存在する場合には、その空間を窒素で置換することなどによって培地への酸素溶解を遮断し、培地中の溶存酸素の細胞による消費量を測定することが必要であった。
しかし、ガス透過性部材からなる培養容器を用いて細胞培養を行う場合は、培地中の細胞が消費する溶存酸素のみの濃度変化を検出することはできず、また培養容器内を撹拌して計測することが必要となるため、静置培養を行うことができないという問題があった。 Furthermore, in the method (3), since it is necessary to measure the amount of decrease in dissolved oxygen in the culture vessel, the gas should not move from the inside of the culture vessel to the outside or the gas should not flow into the culture vessel from the outside. It was necessary to keep it sealed. Alternatively, if there is a space on the medium in the culture medium, oxygen dissolution in the medium should be blocked by replacing the space with nitrogen, and the amount of dissolved oxygen consumed by the cells in the medium should be measured. Was needed.
However, when cell culture is performed using a culture vessel made of a gas permeable member, it is not possible to detect a change in the concentration of only the dissolved oxygen consumed by the cells in the medium, and the inside of the culture vessel is stirred and measured. There was a problem that static culture could not be performed because it was necessary to do so.
しかし、ガス透過性部材からなる培養容器を用いて細胞培養を行う場合は、培地中の細胞が消費する溶存酸素のみの濃度変化を検出することはできず、また培養容器内を撹拌して計測することが必要となるため、静置培養を行うことができないという問題があった。 Furthermore, in the method (3), since it is necessary to measure the amount of decrease in dissolved oxygen in the culture vessel, the gas should not move from the inside of the culture vessel to the outside or the gas should not flow into the culture vessel from the outside. It was necessary to keep it sealed. Alternatively, if there is a space on the medium in the culture medium, oxygen dissolution in the medium should be blocked by replacing the space with nitrogen, and the amount of dissolved oxygen consumed by the cells in the medium should be measured. Was needed.
However, when cell culture is performed using a culture vessel made of a gas permeable member, it is not possible to detect a change in the concentration of only the dissolved oxygen consumed by the cells in the medium, and the inside of the culture vessel is stirred and measured. There was a problem that static culture could not be performed because it was necessary to do so.
ここで、特許文献1には、微生物を含有する試料溶液中の溶存酸素の減少量の測定を、試料溶液を容器内に密閉した状態で行う方法が開示されているが、この測定方法は、ガス透過性のない培養容器を用いて行う必要があった。
また、特許文献2には、培養容器内の培地に溶存する酸素量を測定する酸素量測定手段によって測定された酸素量により、培養容器内の細胞数に関連する酸素量の時間変動量を演算する細胞培養装置が開示されている。しかし、この細胞培養装置は、外部から培養容器内にガスが流入しない密閉容器を使用すると共に、通常の培養状態から計測可能な状態にするために、培養容器内の培地上の空間を窒素で置換して、培地への酸素溶解を遮断するなど培養条件を変化させて培地中の溶存酸素の消費を計測することが必要なものであった。
これらの特許文献に記載の発明は、ガス透過性部材からなる培養容器によって細胞を静置状態で培養する場合に、好適に使用できるものではなかった。 Here, Patent Document 1 discloses a method of measuring the amount of decrease in dissolved oxygen in a sample solution containing a microorganism in a state where the sample solution is sealed in a container. It was necessary to use a culture vessel with no gas permeability.
Further, inPatent Document 2, the amount of time fluctuation of the amount of oxygen related to the number of cells in the culture vessel is calculated from the amount of oxygen measured by the oxygen amount measuring means for measuring the amount of oxygen dissolved in the medium in the culture vessel. The cell culture apparatus to be used is disclosed. However, this cell culture device uses a closed container in which gas does not flow into the culture container from the outside, and in order to make it measurable from the normal culture state, the space on the medium in the culture container is filled with nitrogen. It was necessary to measure the consumption of dissolved oxygen in the medium by substituting and changing the culture conditions such as blocking the dissolution of oxygen in the medium.
The inventions described in these patent documents have not been suitably used when cells are cultured in a static state in a culture vessel made of a gas permeable member.
また、特許文献2には、培養容器内の培地に溶存する酸素量を測定する酸素量測定手段によって測定された酸素量により、培養容器内の細胞数に関連する酸素量の時間変動量を演算する細胞培養装置が開示されている。しかし、この細胞培養装置は、外部から培養容器内にガスが流入しない密閉容器を使用すると共に、通常の培養状態から計測可能な状態にするために、培養容器内の培地上の空間を窒素で置換して、培地への酸素溶解を遮断するなど培養条件を変化させて培地中の溶存酸素の消費を計測することが必要なものであった。
これらの特許文献に記載の発明は、ガス透過性部材からなる培養容器によって細胞を静置状態で培養する場合に、好適に使用できるものではなかった。 Here, Patent Document 1 discloses a method of measuring the amount of decrease in dissolved oxygen in a sample solution containing a microorganism in a state where the sample solution is sealed in a container. It was necessary to use a culture vessel with no gas permeability.
Further, in
The inventions described in these patent documents have not been suitably used when cells are cultured in a static state in a culture vessel made of a gas permeable member.
本発明は、上記事情に鑑みてなされたものであり、ガス透過性部材からなる培養容器によって細胞を静置状態で培養する場合において、培養中の所望のタイミングにおける培養容器内の細胞の増殖性を検出可能な細胞培養システム、及び細胞の増殖性の検出方法の提供を目的とする。
The present invention has been made in view of the above circumstances, and when cells are cultured in a stationary state in a culture vessel made of a gas permeable member, the proliferative potential of the cells in the culture vessel at a desired timing during culture. It is an object of the present invention to provide a cell culture system capable of detecting cells and a method for detecting cell proliferativeness.
上記目的を達成するため、本発明の細胞培養システムは、少なくとも一部がガス透過性部材からなる培養容器によって細胞を静置状態で培養する細胞培養システムであって、前記培養容器内における培養面付近の酸素濃度を計測する酸素濃度センサと、前記ガス透過性部材の酸素透過度、前記酸素濃度センサの測定値、前記培養容器の周囲の酸素濃度、及び前記細胞の1個当たりの酸素消費量にもとづき前記細胞の増殖性を検出する増殖検出部とを有する構成としてある。
In order to achieve the above object, the cell culture system of the present invention is a cell culture system in which cells are cultured in a stationary state by a culture vessel having at least a part of a gas permeable member, and the culture surface in the culture vessel. An oxygen concentration sensor that measures the oxygen concentration in the vicinity, the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption per cell. Based on this, the configuration includes a proliferation detection unit that detects the proliferation of the cells.
また、本発明の細胞培養システムを、前記増殖検出部が、前記細胞の増殖性として、前記細胞の単位面積当たりの個数を、以下の式(1)によって算出する構成とすることが好ましい。
C=G×(M/100-D/100)/S ・・・式(1)
C:細胞の単位面積当たりの個数(個/cm2)
G:ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))
M:培養容器の周囲の酸素濃度(%)
D:酸素濃度センサの測定値(%)
S:細胞1個の単位時間当たりの酸素消費量(mg/(hr・個) ) Further, it is preferable that the cell culture system of the present invention has a configuration in which the proliferation detection unit calculates the number of cells per unit area as the proliferation of the cells by the following formula (1).
C = G × (M / 100-D / 100) / S ... Expression (1)
C: Number of cells per unit area (pieces / cm 2 )
G: Oxygen permeability of gas permeable member (mg / (cm 2・ hr ・ atm))
M: Oxygen concentration around the culture vessel (%)
D: Measured value (%) of oxygen concentration sensor
S: Oxygen consumption per unit time per cell (mg / (hr ·))
C=G×(M/100-D/100)/S ・・・式(1)
C:細胞の単位面積当たりの個数(個/cm2)
G:ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))
M:培養容器の周囲の酸素濃度(%)
D:酸素濃度センサの測定値(%)
S:細胞1個の単位時間当たりの酸素消費量(mg/(hr・個) ) Further, it is preferable that the cell culture system of the present invention has a configuration in which the proliferation detection unit calculates the number of cells per unit area as the proliferation of the cells by the following formula (1).
C = G × (M / 100-D / 100) / S ... Expression (1)
C: Number of cells per unit area (pieces / cm 2 )
G: Oxygen permeability of gas permeable member (mg / (cm 2・ hr ・ atm))
M: Oxygen concentration around the culture vessel (%)
D: Measured value (%) of oxygen concentration sensor
S: Oxygen consumption per unit time per cell (mg / (hr ·))
また、本発明の細胞培養システムを、前記増殖検出部が、前記培養容器に複数回新培地を追加した時の酸素濃度の上昇ピーク値の変化の傾きと前記培養容器の安定時の酸素濃度の変化の傾きとの比率にもとづいて得られる所望の時点の前記培養容器内の酸素濃度の推定値を、前記酸素濃度センサの測定値として用いる構成とすることが好ましい。
Further, in the cell culture system of the present invention, the gradient of the change in the peak value of the increase in oxygen concentration when the growth detection unit added the new medium to the culture vessel a plurality of times and the oxygen concentration when the culture vessel was stabilized. It is preferable to use the estimated value of the oxygen concentration in the culture vessel at a desired time point obtained based on the ratio to the slope of the change as the measured value of the oxygen concentration sensor.
また、本発明の細胞培養システムを、前記培養容器の周囲の酸素濃度が、前記培養容器が収容されたインキュベータ内の酸素濃度、又は、大気中の酸素濃度である構成とすることが好ましい。
Further, it is preferable that the cell culture system of the present invention has a configuration in which the oxygen concentration around the culture vessel is the oxygen concentration in the incubator in which the culture vessel is housed or the oxygen concentration in the atmosphere.
また、本発明の細胞培養システムを、前記酸素濃度センサが、蛍光式である構成とすることが好ましい。
Further, it is preferable that the cell culture system of the present invention has a configuration in which the oxygen concentration sensor is a fluorescent type.
また、本発明の細胞培養システムを、前記酸素濃度センサが、前記培養容器内における培養面に穴あき樹脂部材を用いて固定されている構成とすることが好ましい。
Further, it is preferable that the cell culture system of the present invention has a configuration in which the oxygen concentration sensor is fixed to the culture surface in the culture vessel by using a perforated resin member.
また、本発明の細胞の増殖性の検出方法は、少なくとも一部がガス透過性部材からなる培養容器によって細胞を静置状態で培養する細胞培養における細胞の増殖性の検出方法であって、前記培養容器内における培養面に配置された酸素濃度センサにより前記培養容器内における培養面付近の酸素濃度を計測し、前記ガス透過性部材の酸素透過度、前記酸素濃度センサの測定値、前記培養容器の周囲の酸素濃度、及び前記細胞の1個当たりの酸素消費量にもとづき前記細胞の単位面積当たりの個数を計算する方法としてある。
Further, the method for detecting cell proliferation of the present invention is a method for detecting cell proliferation in a cell culture in which cells are cultured in a stationary state by a culture vessel having at least a part of a gas permeable member. The oxygen concentration near the culture surface in the culture vessel is measured by the oxygen concentration sensor arranged on the culture surface in the culture vessel, and the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, and the culture vessel. There is a method of calculating the number of cells per unit area based on the oxygen concentration around the cells and the amount of oxygen consumed per cell.
本発明によれば、ガス透過性部材からなる培養容器によって細胞を静置状態で培養する場合において、培養中の所望のタイミングにおける培養容器内の細胞の増殖性を検出可能な細胞培養システム、及び細胞の増殖性の検出方法の提供が可能となる。
According to the present invention, when cells are cultured in a stationary state in a culture vessel made of a gas permeable member, a cell culture system capable of detecting the proliferation of cells in the culture vessel at a desired timing during culture, and a cell culture system. It becomes possible to provide a method for detecting cell proliferation.
以下、本発明の細胞培養システム、及び細胞の増殖性の検出方法の実施形態について詳細に説明する。ただし、本発明は、以下の実施形態及び実施例の具体的な内容に限定されるものではない。
Hereinafter, embodiments of the cell culture system of the present invention and the method for detecting cell proliferation will be described in detail. However, the present invention is not limited to the specific contents of the following embodiments and examples.
本実施形態の細胞培養システムは、少なくとも一部がガス透過性部材からなる培養容器によって細胞を静置状態で培養する細胞培養システムであって、培養容器内における培養面付近の酸素濃度を計測する酸素濃度センサと、ガス透過性部材の酸素透過度、酸素濃度センサの測定値、培養容器の周囲の酸素濃度、及び細胞の1個当たりの酸素消費量にもとづき細胞の増殖性を検出する増殖検出部とを有することを特徴とする。
具体的には、本実施形態の細胞培養システムは、例えば図1に示すような構成とすることができる。 The cell culture system of the present embodiment is a cell culture system in which cells are cultured in a stationary state by a culture vessel having at least a part of a gas permeable member, and measures the oxygen concentration near the culture surface in the culture vessel. Proliferation detection that detects cell proliferation based on the oxygen concentration sensor, the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption per cell. It is characterized by having a part.
Specifically, the cell culture system of the present embodiment can be configured as shown in FIG. 1, for example.
具体的には、本実施形態の細胞培養システムは、例えば図1に示すような構成とすることができる。 The cell culture system of the present embodiment is a cell culture system in which cells are cultured in a stationary state by a culture vessel having at least a part of a gas permeable member, and measures the oxygen concentration near the culture surface in the culture vessel. Proliferation detection that detects cell proliferation based on the oxygen concentration sensor, the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption per cell. It is characterized by having a part.
Specifically, the cell culture system of the present embodiment can be configured as shown in FIG. 1, for example.
まず、本実施形態の細胞培養システムの概略構成について説明する。
本実施形態の細胞培養システムにおいて、培養容器20が、インキュベータ10内に収容され載置台11に配置されている。培養容器20には2つのポートが備えられ、一方のポートに保冷庫20に収容されている培地供給容器40がチューブにより連結されている。また、培養容器20の他方のポートにインキュベータ10内の廃液容器50がチューブにより連結されている。 First, the schematic configuration of the cell culture system of the present embodiment will be described.
In the cell culture system of the present embodiment, theculture container 20 is housed in the incubator 10 and arranged on the mounting table 11. The culture container 20 is provided with two ports, and the medium supply container 40 housed in the cold storage 20 is connected to one port by a tube. Further, the waste liquid container 50 in the incubator 10 is connected to the other port of the culture container 20 by a tube.
本実施形態の細胞培養システムにおいて、培養容器20が、インキュベータ10内に収容され載置台11に配置されている。培養容器20には2つのポートが備えられ、一方のポートに保冷庫20に収容されている培地供給容器40がチューブにより連結されている。また、培養容器20の他方のポートにインキュベータ10内の廃液容器50がチューブにより連結されている。 First, the schematic configuration of the cell culture system of the present embodiment will be described.
In the cell culture system of the present embodiment, the
培養容器20と培地供給容器40を連結するチューブ上と、培養容器20と廃液容器50を連結するチューブ上には、送液手段が配設されている。この送液手段としては、例えばペリスタポンプやシリンジポンプのような低速で高精度な送液を行うことが可能なポンプを用いることが好ましい。
Liquid feeding means are arranged on the tube connecting the culture container 20 and the medium supply container 40 and on the tube connecting the culture container 20 and the waste liquid container 50. As the liquid feeding means, it is preferable to use a pump capable of delivering liquid at a low speed and with high accuracy, for example, a perista pump or a syringe pump.
そして、制御装置60によって、所定のタイミングで培養容器20と培地供給容器40間の送液手段が制御され、培地が培地供給容器40から培養容器20へ送液される。また制御装置60によって、所定のタイミングで培養容器20と廃液容器50間の送液手段が制御され、培地が培養容器20から廃液容器50へ送液される。
Then, the control device 60 controls the liquid feeding means between the culture vessel 20 and the culture medium supply container 40 at a predetermined timing, and the culture medium is fed from the culture medium supply container 40 to the culture medium 20. Further, the control device 60 controls the liquid feeding means between the culture container 20 and the waste liquid container 50 at a predetermined timing, and the medium is sent from the culture container 20 to the waste liquid container 50.
インキュベータ10には、酸素ボンベO(酸素供給装置)と二酸化炭素ボンベC(二酸化炭素供給装置)からそれぞれ酸素ガス、二酸化炭素ガスが供給されることにより、その内部のガス濃度が制御されて、培養容器20の周囲のガス濃度を調整することが可能になっている。
Oxygen gas and carbon dioxide gas are supplied to the incubator 10 from the oxygen cylinder O (oxygen supply device) and the carbon dioxide cylinder C (carbon dioxide supply device), respectively, so that the gas concentration inside the incubator 10 is controlled and the culture is performed. It is possible to adjust the gas concentration around the container 20.
すなわち、インキュベータ10には、酸素ボンベOと二酸化炭素ボンベCがそれぞれバルブを介して接続されており、制御装置60の制御によって各バルブの開閉動作が制御され、各ガスボンベからのインキュベータ10へのガスの供給が制御されて、インキュベータ10内の酸素濃度を調整することが可能になっている。
That is, oxygen cylinder O and carbon dioxide cylinder C are connected to the incubator 10 via valves, respectively, and the opening / closing operation of each valve is controlled by the control of the control device 60, and the gas from each gas cylinder to the incubator 10 is controlled. The supply of carbon dioxide is controlled so that the oxygen concentration in the incubator 10 can be adjusted.
インキュベータ10としては、例えばCO2インキュベータなどを用いることができる。インキュベータ10内の載置台11としては、パンチングメタルなどからなるものを設置して用いることができ、載置台11に培養容器20が配置されて閉鎖的に収納される。なお、載置台11は省略してもよい。
本実施形態の細胞培養システムは、インキュベータ10を用いることなく細胞を培養する場合にも適用できるものであるため、インキュベータ10を省略してもよい。 As theincubator 10, for example, a CO2 incubator or the like can be used. As the mounting table 11 in the incubator 10, one made of punching metal or the like can be installed and used, and the culture container 20 is arranged on the mounting table 11 and is stored in a closed manner. The mounting table 11 may be omitted.
Since the cell culture system of the present embodiment can be applied to the case of culturing cells without using theincubator 10, the incubator 10 may be omitted.
本実施形態の細胞培養システムは、インキュベータ10を用いることなく細胞を培養する場合にも適用できるものであるため、インキュベータ10を省略してもよい。 As the
Since the cell culture system of the present embodiment can be applied to the case of culturing cells without using the
次に、本実施形態の細胞培養システムにおける培養容器20について詳細に説明する。
培養容器20は、少なくとも一部がガス透過性部材からなる、ガス透過性を有する閉鎖系の細胞培養容器であり、例えば矩形状の2枚のガス透過性フィルムの周縁部をヒートシールすることによって形成されたものなどを用いることができる。
培養容器20の形状は、ポート付近の形状がポートに対して傾斜して形成され、これによって培地がポートに流れやすくしたものとすることができる。また、長方形などの矩形状とすることもできる。培養容器20に備えられるポートの個数は2個に限定されず、1個又は3個以上であってもよい。 Next, theculture vessel 20 in the cell culture system of the present embodiment will be described in detail.
Theculture vessel 20 is a closed-type cell culture vessel having gas permeability, which is at least partially composed of a gas-permeable member, and is, for example, by heat-sealing the peripheral portions of two rectangular gas-permeable films. The formed one can be used.
The shape of theculture vessel 20 can be formed so that the shape near the port is inclined with respect to the port, whereby the medium can easily flow to the port. It can also be a rectangle such as a rectangle. The number of ports provided in the culture vessel 20 is not limited to two, and may be one or three or more.
培養容器20は、少なくとも一部がガス透過性部材からなる、ガス透過性を有する閉鎖系の細胞培養容器であり、例えば矩形状の2枚のガス透過性フィルムの周縁部をヒートシールすることによって形成されたものなどを用いることができる。
培養容器20の形状は、ポート付近の形状がポートに対して傾斜して形成され、これによって培地がポートに流れやすくしたものとすることができる。また、長方形などの矩形状とすることもできる。培養容器20に備えられるポートの個数は2個に限定されず、1個又は3個以上であってもよい。 Next, the
The
The shape of the
本実施形態の細胞培養システムにおいて、培養容器20内の培養面上には、蛍光式酸素濃度センサ21が配設されている。また、インキュベータ10内において、蛍光式酸素濃度センサ21に対して、蛍光を受発信可能に蛍光受発光部22が配設されている。
蛍光受発光部22は、例えば載置台11の下側に配置され、載置台11が透明な場合にはこれを介して、あるいは載置台11に設けられた貫通孔を介して、蛍光式酸素濃度センサ21に対して蛍光の受発信を行うことができる。なお、蛍光受発光部22を培養容器20の下側に接触して配置させることもできる。
この蛍光受発光部22を制御装置60により制御することで、培養容器20内の酸素濃度を測定することが可能になっている。 In the cell culture system of the present embodiment, the fluorescent oxygen concentration sensor 21 is arranged on the culture surface in theculture container 20. Further, in the incubator 10, a fluorescence receiving / emitting unit 22 is arranged so that fluorescence can be transmitted / received to / from the fluorescent oxygen concentration sensor 21.
The fluorescence receiving / emitting unit 22 is arranged, for example, under the mounting table 11, and if the mounting table 11 is transparent, the fluorescent oxygen concentration is provided through the mounting table 11 or through a through hole provided in the mounting table 11. It is possible to send and receive fluorescence to and from the sensor 21. The fluorescence receiving / emitting unit 22 may be arranged in contact with the lower side of theculture vessel 20.
By controlling the fluorescence receiving / emitting unit 22 by thecontrol device 60, it is possible to measure the oxygen concentration in the culture vessel 20.
蛍光受発光部22は、例えば載置台11の下側に配置され、載置台11が透明な場合にはこれを介して、あるいは載置台11に設けられた貫通孔を介して、蛍光式酸素濃度センサ21に対して蛍光の受発信を行うことができる。なお、蛍光受発光部22を培養容器20の下側に接触して配置させることもできる。
この蛍光受発光部22を制御装置60により制御することで、培養容器20内の酸素濃度を測定することが可能になっている。 In the cell culture system of the present embodiment, the fluorescent oxygen concentration sensor 21 is arranged on the culture surface in the
The fluorescence receiving / emitting unit 22 is arranged, for example, under the mounting table 11, and if the mounting table 11 is transparent, the fluorescent oxygen concentration is provided through the mounting table 11 or through a through hole provided in the mounting table 11. It is possible to send and receive fluorescence to and from the sensor 21. The fluorescence receiving / emitting unit 22 may be arranged in contact with the lower side of the
By controlling the fluorescence receiving / emitting unit 22 by the
図2に示すように、培養容器20内の培地における溶存酸素濃度は、場所によって大きく異なっている。すなわち、細胞が存在する培養面(下面ガス透過性フィルム202の上面)付近の培地では、細胞による酸素消費が活発に行われるため、酸素が急速に消費され、細胞密度が増大するにしたがって、次第に0%になっていく。これに対して、培養容器20内の液厚にもよるが、例えば液厚が10mm以上の場合には、培養面の反対側の培養容器20内の上面(上面ガス透過性フィルム201の下面)付近の培地では、細胞が近辺に存在していない場合、酸素濃度は初期の状態(例えば大気中の約21%)からなかなか減少していかない。培養容器20の中央付近の培地では、これらの中間程度の酸素濃度(例えば10%)になっている。
As shown in FIG. 2, the dissolved oxygen concentration in the medium in the culture vessel 20 varies greatly depending on the location. That is, in the medium near the culture surface (upper surface of the lower gas permeable film 202) where the cells are present, oxygen is actively consumed by the cells, so that oxygen is rapidly consumed and gradually increases as the cell density increases. It becomes 0%. On the other hand, although it depends on the liquid thickness in the culture container 20, for example, when the liquid thickness is 10 mm or more, the upper surface in the culture container 20 on the opposite side of the culture surface (lower surface of the upper surface gas permeable film 201). In the nearby medium, the oxygen concentration does not decrease easily from the initial state (for example, about 21% in the atmosphere) when the cells are not present in the vicinity. In the medium near the center of the culture vessel 20, the oxygen concentration is in the middle of these (for example, 10%).
したがって、本実施形態の細胞培養システムでは、蛍光式酸素濃度センサ21を培養容器20内の培養面に配置させることによって、細胞による酸素消費にもとづく培養容器20内の酸素濃度を適切に計測することを可能にしている。
また、後述するように、本実施形態の細胞培養システムでは、培養容器20の酸素透過度と、培養容器20の周囲の酸素濃度を加味することによって、細胞による酸素の消費量を適切に算出することを可能にしている。
さらに、本実施形態の細胞培養システムによれば、培養容器20内の酸素濃度が0%~1%程度に継続してなり、蛍光式酸素濃度センサ21による酸素濃度の計測が困難な場合についても、細胞による酸素の消費量を適切に推定することを可能にしている。 Therefore, in the cell culture system of the present embodiment, by arranging the fluorescent oxygen concentration sensor 21 on the culture surface in theculture vessel 20, the oxygen concentration in the culture vessel 20 based on the oxygen consumption by the cells is appropriately measured. Is possible.
Further, as will be described later, in the cell culture system of the present embodiment, the oxygen consumption by the cells is appropriately calculated by adding the oxygen permeability of theculture vessel 20 and the oxygen concentration around the culture vessel 20. It makes it possible.
Further, according to the cell culture system of the present embodiment, even when the oxygen concentration in theculture vessel 20 continues to be about 0% to 1% and it is difficult to measure the oxygen concentration by the fluorescent oxygen concentration sensor 21. It makes it possible to properly estimate the amount of oxygen consumed by cells.
また、後述するように、本実施形態の細胞培養システムでは、培養容器20の酸素透過度と、培養容器20の周囲の酸素濃度を加味することによって、細胞による酸素の消費量を適切に算出することを可能にしている。
さらに、本実施形態の細胞培養システムによれば、培養容器20内の酸素濃度が0%~1%程度に継続してなり、蛍光式酸素濃度センサ21による酸素濃度の計測が困難な場合についても、細胞による酸素の消費量を適切に推定することを可能にしている。 Therefore, in the cell culture system of the present embodiment, by arranging the fluorescent oxygen concentration sensor 21 on the culture surface in the
Further, as will be described later, in the cell culture system of the present embodiment, the oxygen consumption by the cells is appropriately calculated by adding the oxygen permeability of the
Further, according to the cell culture system of the present embodiment, even when the oxygen concentration in the
ここで、ガス透過性フィルムにおけるガス透過のメカニズムについて説明する。
培養容器内の培地中の溶存ガスは、培養容器の周囲の分圧に平衡となる濃度で溶解している。インキュベータに置かれた培地に溶解している酸素の濃度は、インキュベータ内のガス濃度の制御を行っていない場合、大気中の濃度と同じで約21%である。培養容器内の溶存酸素を細胞が消費すると細胞周辺の培地の溶存酸素が減少して、培養容器の外側の酸素濃度と平衡を保つように酸素ガスがフィルムを透過して培養容器内に流入する。
フィルムのガス透過性(速度)は、フィルム両側のガス分圧差、フィルムとガスの親和性、及びフィルム中にガス分子が移動する拡散性などによって決定される。 Here, the mechanism of gas permeation in the gas permeation film will be described.
The dissolved gas in the medium in the culture vessel is dissolved at a concentration that is in equilibrium with the partial pressure around the culture vessel. The concentration of oxygen dissolved in the medium placed in the incubator is about 21%, which is the same as the concentration in the atmosphere when the gas concentration in the incubator is not controlled. When cells consume the dissolved oxygen in the culture vessel, the dissolved oxygen in the medium around the cells decreases, and oxygen gas permeates the film and flows into the culture vessel so as to maintain equilibrium with the oxygen concentration outside the culture vessel. ..
The gas permeability (velocity) of a film is determined by the difference in gas partial pressure between both sides of the film, the affinity between the film and the gas, and the diffusivity of moving gas molecules into the film.
培養容器内の培地中の溶存ガスは、培養容器の周囲の分圧に平衡となる濃度で溶解している。インキュベータに置かれた培地に溶解している酸素の濃度は、インキュベータ内のガス濃度の制御を行っていない場合、大気中の濃度と同じで約21%である。培養容器内の溶存酸素を細胞が消費すると細胞周辺の培地の溶存酸素が減少して、培養容器の外側の酸素濃度と平衡を保つように酸素ガスがフィルムを透過して培養容器内に流入する。
フィルムのガス透過性(速度)は、フィルム両側のガス分圧差、フィルムとガスの親和性、及びフィルム中にガス分子が移動する拡散性などによって決定される。 Here, the mechanism of gas permeation in the gas permeation film will be described.
The dissolved gas in the medium in the culture vessel is dissolved at a concentration that is in equilibrium with the partial pressure around the culture vessel. The concentration of oxygen dissolved in the medium placed in the incubator is about 21%, which is the same as the concentration in the atmosphere when the gas concentration in the incubator is not controlled. When cells consume the dissolved oxygen in the culture vessel, the dissolved oxygen in the medium around the cells decreases, and oxygen gas permeates the film and flows into the culture vessel so as to maintain equilibrium with the oxygen concentration outside the culture vessel. ..
The gas permeability (velocity) of a film is determined by the difference in gas partial pressure between both sides of the film, the affinity between the film and the gas, and the diffusivity of moving gas molecules into the film.
次に、蛍光式酸素濃度センサによる酸素濃度の測定原理について説明する。
蛍光式酸素濃度センサの蛍光素子に光を当てると、蛍光物質は光を吸収して励起状態となり、蛍光を放射しながら基底状態に戻る。このとき、蛍光物質の周りに酸素があると励起エネルギーが奪われて蛍光発光強度が下がる。したがって、酸素が多いほど蛍光発光強度が小さくなる。蛍光受発光部は、培地に溶存している酸素が蛍光素子に接触し、その接触している酸素分子の数による発光強度の変化にもとづき酸素の量を測定している。 Next, the principle of measuring the oxygen concentration by the fluorescent oxygen concentration sensor will be described.
When light is applied to the fluorescent element of the fluorescent oxygen concentration sensor, the fluorescent substance absorbs the light and becomes an excited state, and returns to the ground state while radiating fluorescence. At this time, if oxygen is present around the fluorescent substance, the excitation energy is deprived and the fluorescence emission intensity is lowered. Therefore, the more oxygen there is, the smaller the fluorescence emission intensity. The fluorescence receiving / emitting unit measures the amount of oxygen based on the change in emission intensity depending on the number of oxygen molecules in contact with the oxygen molecule in contact with the oxygen dissolved in the medium.
蛍光式酸素濃度センサの蛍光素子に光を当てると、蛍光物質は光を吸収して励起状態となり、蛍光を放射しながら基底状態に戻る。このとき、蛍光物質の周りに酸素があると励起エネルギーが奪われて蛍光発光強度が下がる。したがって、酸素が多いほど蛍光発光強度が小さくなる。蛍光受発光部は、培地に溶存している酸素が蛍光素子に接触し、その接触している酸素分子の数による発光強度の変化にもとづき酸素の量を測定している。 Next, the principle of measuring the oxygen concentration by the fluorescent oxygen concentration sensor will be described.
When light is applied to the fluorescent element of the fluorescent oxygen concentration sensor, the fluorescent substance absorbs the light and becomes an excited state, and returns to the ground state while radiating fluorescence. At this time, if oxygen is present around the fluorescent substance, the excitation energy is deprived and the fluorescence emission intensity is lowered. Therefore, the more oxygen there is, the smaller the fluorescence emission intensity. The fluorescence receiving / emitting unit measures the amount of oxygen based on the change in emission intensity depending on the number of oxygen molecules in contact with the oxygen molecule in contact with the oxygen dissolved in the medium.
培養容器20の材料としては、樹脂フィルムなどを好適に用いることができ、ポリエチレンやポリプロピレンなどのポリオレフィン系樹脂等を用いることができる。例えば、ポリエチレン、エチレンとα-オレフィンの共重合体、エチレンと酢酸ビニルの共重合体、エチレンとアクリル酸やメタクリル酸共重合体と金属イオンを用いたアイオノマー等を挙げることができる。また、ポリオレフィン、スチレン系エラストマー、ポリエステル系熱可塑性エラストマー等を用いることもできる。さらに、軟質塩化ビニル樹脂、ポリブタジエン樹脂、エチレン-酢酸ビニル共重合体、塩素化ポリエチレン樹脂、ポリウレタン系熱可塑性エラストマー、ポリエステル系熱可塑性エラストマー、シリコーン系熱可塑性エラストマー、スチレン系エラストマー、例えば、SBS(スチレン・ブタジエン・スチレン)、SIS(スチレン・イソプレン・スチレン)、SEBS(スチレン・エチレン・ブチレン・スチレン)、SEPS(スチレン・エチレン・プロピレン・スチレン)、ポリオレフィン樹脂、フッ素系樹脂等を用いてもよい。
As the material of the culture container 20, a resin film or the like can be preferably used, and a polyolefin-based resin such as polyethylene or polypropylene can be used. For example, polyethylene, a copolymer of ethylene and α-olefin, a copolymer of ethylene and vinyl acetate, an ionomer using ethylene and acrylic acid, a methacrylic acid copolymer and metal ions, and the like can be mentioned. Further, polyolefins, styrene-based elastomers, polyester-based thermoplastic elastomers and the like can also be used. Further, soft vinyl chloride resin, polybutadiene resin, ethylene-vinyl acetate copolymer, chlorinated polyethylene resin, polyurethane-based thermoplastic elastomer, polyester-based thermoplastic elastomer, silicone-based thermoplastic elastomer, styrene-based elastomer, for example, SBS (styrene). -Butadiene / styrene), SIS (styrene / isoprene / styrene), SEBS (styrene / ethylene / butylene / styrene), SEPS (styrene / ethylene / propylene / styrene), polyolefin resin, fluororesin and the like may be used.
また、培養容器20の少なくとも一部に用いられるガス透過性部材としては、上記材料のうち、特にガス透過性能に優れた熱可塑性樹脂を用いることが好ましく、例えばLLDPEなどのポリエチレンやポリプロピレン等のポリオレフィン系樹脂を好適に用いることができる。また、ガス透過性部材は、透明材であることが好ましい。
Further, as the gas permeable member used for at least a part of the culture vessel 20, it is preferable to use a thermoplastic resin having particularly excellent gas permeable performance among the above materials, for example, a polyolefin such as polyethylene such as LLDPE or polypropylene. A based resin can be preferably used. Further, the gas permeable member is preferably a transparent material.
また、図3に示すように、本実施形態の細胞培養システムの変形例として、蛍光式酸素濃度センサ21aが、培養容器20a内の培養面(同図の例では下面ガス透過性フィルム202aの上面)に穴あき樹脂部材23aを用いて固定されていることが好ましい。このとき、穴あき樹脂部材23aと培養面の間に蛍光式酸素濃度センサ21aに挟み込み、穴あき樹脂部材23aの溶着領域Hを培養面に溶着させて固定させることができる。
Further, as shown in FIG. 3, as a modification of the cell culture system of the present embodiment, the fluorescent oxygen concentration sensor 21a is used as a culture surface in the culture container 20a (in the example of the figure, the upper surface of the lower surface gas permeable film 202a). ) Is preferably fixed using a perforated resin member 23a. At this time, the fluorescent oxygen concentration sensor 21a is sandwiched between the perforated resin member 23a and the culture surface, and the welding region H of the perforated resin member 23a can be welded and fixed to the culture surface.
穴あき樹脂部材23aの材料としては、例えばLLDPEなどのポリエチレンやポリプロピレン等のポリオレフィン系樹脂を好適に用いることができる。例えば、ポリエチレン、エチレンとα-オレフィンの共重合体、エチレンと酢酸ビニルの共重合体、エチレンとアクリル酸やメタクリル酸共重合体と金属イオンを用いたアイオノマー等を挙げることができる。
As the material of the perforated resin member 23a, for example, a polyolefin resin such as polyethylene such as LLDPE or polypropylene can be preferably used. For example, polyethylene, a copolymer of ethylene and α-olefin, a copolymer of ethylene and vinyl acetate, an ionomer using ethylene and acrylic acid, a methacrylic acid copolymer and metal ions, and the like can be mentioned.
穴あき樹脂部材23aは、蛍光式酸素濃度センサ21aを培養面に固定しつつ、蛍光式酸素濃度センサ21aを培地に接触させるものであるため、メッシュ状材料を好適に用いることができる。また、穴あき樹脂部材23aとして、例えばスポンジのような多孔質材を用いることもできる。
Since the perforated resin member 23a is for contacting the fluorescent oxygen concentration sensor 21a with the medium while fixing the fluorescent oxygen concentration sensor 21a to the culture surface, a mesh-like material can be preferably used. Further, as the perforated resin member 23a, a porous material such as a sponge can also be used.
具体的なメッシュ状材料及び多孔質材としては、特に限定されないが、培養容器20aの材料と融点が近く、材質が同様のものが好ましく、例えばポリエチレン製メッシュ材(株式会社NBCメッシュテック,PE 200目等)やポリエチレン製多孔質(帝人株式会社,ミライム 大口径≦3μm)などを好適に用いることができる。
The specific mesh-like material and porous material are not particularly limited, but those having a melting point close to that of the culture vessel 20a and the same material are preferable, for example, a polyethylene mesh material (NBC Mesh Tech Co., Ltd., PE200). (Eyes, etc.) and polyethylene porous (Teijin Co., Ltd., Milime, large diameter ≤ 3 μm) can be preferably used.
本実施形態の細胞培養システムを、このように穴あき樹脂部材23aを用いて蛍光式酸素濃度センサ21aが固定された構成とすることによって、培養容器20aへ培地を注入したり、培養容器20aから培地を排出したりする場合などに蛍光式酸素濃度センサ21aが外れて移動することを防止することが可能である。
By configuring the cell culture system of the present embodiment in which the fluorescent oxygen concentration sensor 21a is fixed by using the perforated resin member 23a in this way, the medium can be injected into the culture vessel 20a or from the culture vessel 20a. It is possible to prevent the fluorescent oxygen concentration sensor 21a from coming off and moving when the medium is discharged.
培養容器20を用いて培養する細胞は、特に限定されず、培養液中に浮遊させて培養が行われるリンパ球や樹状細胞などの浮遊性細胞であっても、培養容器内の培養面に接着させて培養が行われる人工多能性幹細胞(iPS細胞)、神経幹細胞、胚性幹細胞(ES細胞)、間葉系幹細胞、肝細胞、膵島細胞、心筋細胞、角膜内皮細胞、及び活性化工程のリンパ球等の接着性細胞であってもよい。
The cells to be cultured using the culture vessel 20 are not particularly limited, and even floating cells such as lymphocytes and dendritic cells that are suspended in the culture medium and cultured can be placed on the culture surface in the culture vessel. Artificial pluripotent stem cells (iPS cells), neural stem cells, embryonic stem cells (ES cells), mesenchymal stem cells, hepatocytes, pancreatic islet cells, myocardial cells, corneal endothelial cells, and activation steps that are adhered and cultured. It may be an adhesive cell such as a lymphocyte.
また、培養容器20を用いて培養する細胞は、スフェロイドやオルガノイドなどの凝集塊を形成させたものであってもよく、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法は、凝集塊の培養にも適用することが可能である。
Further, the cells to be cultured using the culture vessel 20 may be those in which aggregates such as spheroids and organoids are formed, and the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells are aggregated. It can also be applied to mass culture.
次に、本実施形態の細胞培養システムにおける制御装置60について詳細に説明する。
制御装置60(制御ユニット)は、図1に示すように、入出力部61、蛍光式センサ入出力部62、制御部63、操作部64、及び電源部65を有している。 Next, thecontrol device 60 in the cell culture system of the present embodiment will be described in detail.
As shown in FIG. 1, the control device 60 (control unit) has an input /output unit 61, a fluorescent sensor input / output unit 62, a control unit 63, an operation unit 64, and a power supply unit 65.
制御装置60(制御ユニット)は、図1に示すように、入出力部61、蛍光式センサ入出力部62、制御部63、操作部64、及び電源部65を有している。 Next, the
As shown in FIG. 1, the control device 60 (control unit) has an input /
入出力部61は、培養容器20と培地供給容器40の間に配置されたポンプと、培養容器20と廃液容器50の間に配置されたポンプに接続され、制御部63からの入力情報にもとづいて、これらのポンプの動作を制御する。これによって、培養容器20に培地供給容器40から培地が供給され、また培養容器20から培地が廃液容器50に排出される。
The input / output unit 61 is connected to a pump arranged between the culture container 20 and the medium supply container 40 and a pump arranged between the culture container 20 and the waste liquid container 50, and is based on the input information from the control unit 63. And control the operation of these pumps. As a result, the medium is supplied to the culture container 20 from the medium supply container 40, and the medium is discharged from the culture container 20 to the waste liquid container 50.
また、入出力部61は、インキュベータ10のガスセンサ12と、酸素ボンベOとインキュベータ10の間に配置されたバルブと、二酸化炭素ボンベCとインキュベータ10の間に配置されたバルブに接続され、ガスセンサ12からの入力情報を制御部63へ送信する。また、入出力部61は、制御部63からの入力情報にもとづいて、これらのバルブの動作を制御する。これによって、酸素ボンベOから酸素ガスがインキュベータ10内に供給され、また二酸化炭素ボンベCから二酸化炭素ガスをインキュベータ10内に供給することができ、インキュベータ10内のガス濃度を所望のレベルに制御することが可能になっている。
Further, the input / output unit 61 is connected to the gas sensor 12 of the incubator 10, the valve arranged between the oxygen cylinder O and the incubator 10, and the valve arranged between the carbon dioxide cylinder C and the incubator 10, and the gas sensor 12 is connected. The input information from is transmitted to the control unit 63. Further, the input / output unit 61 controls the operation of these valves based on the input information from the control unit 63. Thereby, oxygen gas can be supplied into the incubator 10 from the oxygen cylinder O, and carbon dioxide gas can be supplied into the incubator 10 from the carbon dioxide cylinder C, and the gas concentration in the incubator 10 can be controlled to a desired level. Is possible.
蛍光式センサ入出力部62は、蛍光受発光部22に接続され、制御部63からの入力情報にもとづき蛍光受発光部22による蛍光の発光を制御すると共に、蛍光式酸素濃度センサ21からの受光にもとづく情報を制御部63へ送信する。これによって、培養容器20内の培養面付近の酸素濃度を計測することができ、制御部63は、この酸素濃度を細胞の増殖性の検出に用いることが可能になっている。
The fluorescence sensor input / output unit 62 is connected to the fluorescence light receiving / emitting unit 22, controls the emission of fluorescence by the fluorescence receiving / emitting unit 22 based on the input information from the control unit 63, and receives light from the fluorescent oxygen concentration sensor 21. Information based on this is transmitted to the control unit 63. As a result, the oxygen concentration in the vicinity of the culture surface in the culture vessel 20 can be measured, and the control unit 63 can use this oxygen concentration for detecting the proliferativeness of the cells.
制御部63は、PLC(programmable logic controller,プログラマブルロジックコントローラ)などにより構成され、所望の制御内容を予めプログラミングして記憶させ、これにもとづいて各部の動作を制御することができる。
すなわち、制御部63は、ポンプやバルブを制御するための情報を所定のタイミングで入出力部61に送信する。また、蛍光式センサ入出力部62からの入力情報、時間情報、その他の各種情報にもとづいて、ポンプやバルブを制御するための情報を入出力部61に送信するようにすることもできる。 The control unit 63 is configured by a PLC (programmable logic controller) or the like, and can pre-program and store desired control contents, and can control the operation of each unit based on this.
That is, the control unit 63 transmits information for controlling the pump or valve to the input /output unit 61 at a predetermined timing. Further, it is also possible to transmit information for controlling the pump or valve to the input / output unit 61 based on the input information from the fluorescent sensor input / output unit 62, the time information, and various other information.
すなわち、制御部63は、ポンプやバルブを制御するための情報を所定のタイミングで入出力部61に送信する。また、蛍光式センサ入出力部62からの入力情報、時間情報、その他の各種情報にもとづいて、ポンプやバルブを制御するための情報を入出力部61に送信するようにすることもできる。 The control unit 63 is configured by a PLC (programmable logic controller) or the like, and can pre-program and store desired control contents, and can control the operation of each unit based on this.
That is, the control unit 63 transmits information for controlling the pump or valve to the input /
また、制御部63は、培養容器20内の細胞の増殖性を検出する増殖検出部631を備えている。
増殖検出部631は、ガス透過性部材の酸素透過度、酸素濃度センサの測定値、培養容器の周囲の酸素濃度、及び細胞の1個当たりの酸素消費量にもとづいて、細胞の増殖性を検出する。具体的には、増殖検出部631は、細胞の増殖性として、細胞の単位面積当たりの個数を算出することができる。 Further, the control unit 63 includes agrowth detection unit 631 for detecting the proliferation of cells in the culture vessel 20.
Theproliferation detection unit 631 detects the proliferation of cells based on the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption per cell. do. Specifically, the proliferation detection unit 631 can calculate the number of cells per unit area as the proliferation of cells.
増殖検出部631は、ガス透過性部材の酸素透過度、酸素濃度センサの測定値、培養容器の周囲の酸素濃度、及び細胞の1個当たりの酸素消費量にもとづいて、細胞の増殖性を検出する。具体的には、増殖検出部631は、細胞の増殖性として、細胞の単位面積当たりの個数を算出することができる。 Further, the control unit 63 includes a
The
ガス透過性部材の酸素透過度、及び細胞の1個当たりの酸素消費量については、用途に応じて事前に算出し、増殖検出部631などに記憶させておく。
後述するように、PBMCの細胞1個の酸素消費量を算出した結果、1.0e-9mg/(hr・個)であった。また、Jurkat(リンパ球株化細胞)の細胞1個の酸素消費量を算出した結果、1.74e-9mg/(hr・個)であった。 The oxygen permeability of the gas permeable member and the oxygen consumption per cell are calculated in advance according to the intended use and stored in thegrowth detection unit 631 or the like.
As will be described later, the oxygen consumption of one PBMC cell was calculated to be 1.0e-9 mg / (hr ·). In addition, as a result of calculating the oxygen consumption of one Jurkat (lymphocyte-strained cell) cell, it was 1.74e-9 mg / (hr · cell).
後述するように、PBMCの細胞1個の酸素消費量を算出した結果、1.0e-9mg/(hr・個)であった。また、Jurkat(リンパ球株化細胞)の細胞1個の酸素消費量を算出した結果、1.74e-9mg/(hr・個)であった。 The oxygen permeability of the gas permeable member and the oxygen consumption per cell are calculated in advance according to the intended use and stored in the
As will be described later, the oxygen consumption of one PBMC cell was calculated to be 1.0e-9 mg / (hr ·). In addition, as a result of calculating the oxygen consumption of one Jurkat (lymphocyte-strained cell) cell, it was 1.74e-9 mg / (hr · cell).
酸素透過度(mg/(cm2・hr・atm))の決定に先だって、まず実際の培養形態を決定する。すなわち、酸素透過度は、容器サイズ、充填培地量、蛍光式センサ設置位置、押圧治具の使用の有無を決定した上で、以下のように算出することができる。
Prior to determining the oxygen permeability (mg / (cm 2 · hr · atm)), the actual culture form is first determined. That is, the oxygen permeability can be calculated as follows after determining the container size, the amount of the filling medium, the position where the fluorescent sensor is installed, and whether or not the pressing jig is used.
例えば、培養容器20として、厚みが110μmのポリエチレンフィルム2枚貼り合わせた底面積50cm2の後述する試験1における培養バッグを用いて、この培養バッグ内面のほぼ中央に蛍光式酸素濃度センサ21を固定し、培養容器20を押圧治具に挟み込んで加圧した状態で培養を行うとする。押圧治具の底面は透明の樹脂板であり、樹脂板における蛍光式酸素濃度センサ21に対応する位置に蛍光受発光部22を固定する。
For example, as the culture container 20, the culture bag in Test 1 described later having a bottom area of 50 cm 2 in which two polyethylene films having a thickness of 110 μm are bonded is used, and the fluorescent oxygen concentration sensor 21 is fixed to substantially the center of the inner surface of the culture bag. Then, it is assumed that the culture vessel 20 is sandwiched between the pressing jigs and the culture is performed in a pressurized state. The bottom surface of the pressing jig is a transparent resin plate, and the fluorescence receiving / emitting unit 22 is fixed at a position corresponding to the fluorescent oxygen concentration sensor 21 on the resin plate.
次いで培地を培養温度である37℃に加温して、細胞と共に培養容器20に注入する。細胞として、PBMC(Peripheral Blood Mononuclear Cells,末梢血単核細胞)とJurkat(リンパ球株化細胞)の2種類を使用し、それぞれ2回酸素濃度の変化量の測定を行って、以下のように酸素透過度を算出し、その平均値を得た。
PBMCの培地としては、AlyS505N-7(株式会社細胞科学研究所製)にFBS2%を添加したものを使用し、培養バッグ内に35mlの培地を細胞と共に充填した。また、Jurkat培地としては、AlyS505N-0(株式会社細胞科学研究所製)にFBS2%を添加したものを使用し、培養バッグ内に35mlの培地を細胞と共に充填した。 The medium is then heated to the culture temperature of 37 ° C. and injected into theculture vessel 20 together with the cells. Two types of cells, PBMC (Peripheral Blood Mononuclear Cells) and Jurkat (Lymphocytic cells), were used, and the amount of change in oxygen concentration was measured twice for each, as shown below. Oxygen permeability was calculated and the average value was obtained.
As the medium for PBMC, AlyS505N-7 (manufactured by Cell Science Laboratory Co., Ltd.) to which 2% of FBS was added was used, and 35 ml of the medium was filled in the culture bag together with the cells. As the Jurkat medium, AlyS505N-0 (manufactured by Cell Science Laboratory Co., Ltd.) to whichFBS 2% was added was used, and 35 ml of the medium was filled in the culture bag together with the cells.
PBMCの培地としては、AlyS505N-7(株式会社細胞科学研究所製)にFBS2%を添加したものを使用し、培養バッグ内に35mlの培地を細胞と共に充填した。また、Jurkat培地としては、AlyS505N-0(株式会社細胞科学研究所製)にFBS2%を添加したものを使用し、培養バッグ内に35mlの培地を細胞と共に充填した。 The medium is then heated to the culture temperature of 37 ° C. and injected into the
As the medium for PBMC, AlyS505N-7 (manufactured by Cell Science Laboratory Co., Ltd.) to which 2% of FBS was added was used, and 35 ml of the medium was filled in the culture bag together with the cells. As the Jurkat medium, AlyS505N-0 (manufactured by Cell Science Laboratory Co., Ltd.) to which
PBMCを単位面積当たりの細胞充填量を2.1e6個/cm2として、上記培養バッグに充填後、概ね2時間後に細胞が沈み、酸素濃度が安定した。その際の酸素濃度は、6%であった。
この場合、酸素透過度Gは、以下のように算出することができる。
G=単位面積当たりの細胞充填量×細胞1個の酸素消費量/酸素濃度の変化量
=2.1e6×1.0e-9/(21/100-6/100)
=1.40e-2(mg/(cm2・hr・atm)) The cell filling amount per unit area of PBMC was 2.1 e6 cells / cm 2 , and the cells sank approximately 2 hours after filling the culture bag, and the oxygen concentration became stable. The oxygen concentration at that time was 6%.
In this case, the oxygen permeability G can be calculated as follows.
G = Cell filling amount per unit area x Oxygen consumption per cell / Change in oxygen concentration = 2.1e6 x 1.0e-9 / (21 / 100-6 / 100)
= 1.40e-2 (mg / (cm 2・ hr ・ atm))
この場合、酸素透過度Gは、以下のように算出することができる。
G=単位面積当たりの細胞充填量×細胞1個の酸素消費量/酸素濃度の変化量
=2.1e6×1.0e-9/(21/100-6/100)
=1.40e-2(mg/(cm2・hr・atm)) The cell filling amount per unit area of PBMC was 2.1 e6 cells / cm 2 , and the cells sank approximately 2 hours after filling the culture bag, and the oxygen concentration became stable. The oxygen concentration at that time was 6%.
In this case, the oxygen permeability G can be calculated as follows.
G = Cell filling amount per unit area x Oxygen consumption per cell / Change in oxygen concentration = 2.1e6 x 1.0e-9 / (21 / 100-6 / 100)
= 1.40e-2 (mg / (cm 2・ hr ・ atm))
PBMCを単位面積当たりの細胞充填量を4.0e6個/cm2として、上記培養バッグに充填後、培養バッグの周囲の酸素濃度を30%に制御した。概ね2時間後に細胞が沈み、酸素濃度が安定した。その際の酸素濃度は、2%であった。
この場合、酸素透過度Gは、以下のように算出することができる。
G=単位面積当たりの細胞充填量×細胞1個の酸素消費量/酸素濃度の変化量
=4.0e6×1.0e-9/(30/100-2/100)
=1.43e-2(mg/(cm2・hr・atm)) After filling the culture bag with PBMC at a cell filling amount of 4.0 e6 cells / cm 2 per unit area, the oxygen concentration around the culture bag was controlled to 30%. After about 2 hours, the cells sank and the oxygen concentration became stable. The oxygen concentration at that time was 2%.
In this case, the oxygen permeability G can be calculated as follows.
G = Cell filling amount per unit area x Oxygen consumption per cell / Change in oxygen concentration = 4.0e6 x 1.0e-9 / (30 / 100-2 / 100)
= 1.43e-2 (mg / (cm 2・ hr ・ atm))
この場合、酸素透過度Gは、以下のように算出することができる。
G=単位面積当たりの細胞充填量×細胞1個の酸素消費量/酸素濃度の変化量
=4.0e6×1.0e-9/(30/100-2/100)
=1.43e-2(mg/(cm2・hr・atm)) After filling the culture bag with PBMC at a cell filling amount of 4.0 e6 cells / cm 2 per unit area, the oxygen concentration around the culture bag was controlled to 30%. After about 2 hours, the cells sank and the oxygen concentration became stable. The oxygen concentration at that time was 2%.
In this case, the oxygen permeability G can be calculated as follows.
G = Cell filling amount per unit area x Oxygen consumption per cell / Change in oxygen concentration = 4.0e6 x 1.0e-9 / (30 / 100-2 / 100)
= 1.43e-2 (mg / (cm 2・ hr ・ atm))
Jurkatを単位面積当たりの細胞充填量を1.1e6個/cm2として、上記培養バッグに充填後、概ね2時間後に細胞が沈み、酸素濃度が安定する。その際の酸素濃度は、8%であった。
この場合、酸素透過度Gは、以下のように算出することができる。
G=単位面積当たりの細胞充填量×細胞1個の酸素消費量/酸素濃度の変化量
=1.1e6×1.74e-9/(21/100-8/100)
=1.47e-2(mg/(cm2・hr・atm)) With Jurkat as the cell filling amount per unit area of 1.1 e6 cells / cm 2 , the cells sink approximately 2 hours after filling in the culture bag, and the oxygen concentration stabilizes. The oxygen concentration at that time was 8%.
In this case, the oxygen permeability G can be calculated as follows.
G = Cell filling amount per unit area x Oxygen consumption per cell / Change in oxygen concentration = 1.1e6 x 1.74e-9 / (21 / 100-8 / 100)
= 1.47e-2 (mg / (cm 2・ hr ・ atm))
この場合、酸素透過度Gは、以下のように算出することができる。
G=単位面積当たりの細胞充填量×細胞1個の酸素消費量/酸素濃度の変化量
=1.1e6×1.74e-9/(21/100-8/100)
=1.47e-2(mg/(cm2・hr・atm)) With Jurkat as the cell filling amount per unit area of 1.1 e6 cells / cm 2 , the cells sink approximately 2 hours after filling in the culture bag, and the oxygen concentration stabilizes. The oxygen concentration at that time was 8%.
In this case, the oxygen permeability G can be calculated as follows.
G = Cell filling amount per unit area x Oxygen consumption per cell / Change in oxygen concentration = 1.1e6 x 1.74e-9 / (21 / 100-8 / 100)
= 1.47e-2 (mg / (cm 2・ hr ・ atm))
Jurkatを単位面積当たりの細胞充填量を1.9e6個/cm2として、上記培養バッグに充填後、培養バッグの周囲の酸素濃度を30%に制御した。概ね2時間後に細胞が沈み、酸素濃度が安定した。その際の酸素濃度は、5%であった。
この場合、酸素透過度Gは、以下のように算出することができる。
G=単位面積当たりの細胞充填量×細胞1個の酸素消費量/酸素濃度の変化量
=1.9e6×1.74e-9/(30/100-5/100)
=1.32e-2(mg/(cm2・hr・atm)) The cell filling amount per unit area of Jurkat was 1.9 e6 cells / cm 2 , and after filling the culture bag, the oxygen concentration around the culture bag was controlled to 30%. After about 2 hours, the cells sank and the oxygen concentration became stable. The oxygen concentration at that time was 5%.
In this case, the oxygen permeability G can be calculated as follows.
G = Cell filling amount per unit area x Oxygen consumption per cell / Change in oxygen concentration = 1.9e6 x 1.74e-9 / (30 / 100-5 / 100)
= 1.32e-2 (mg / (cm 2・ hr ・ atm))
この場合、酸素透過度Gは、以下のように算出することができる。
G=単位面積当たりの細胞充填量×細胞1個の酸素消費量/酸素濃度の変化量
=1.9e6×1.74e-9/(30/100-5/100)
=1.32e-2(mg/(cm2・hr・atm)) The cell filling amount per unit area of Jurkat was 1.9 e6 cells / cm 2 , and after filling the culture bag, the oxygen concentration around the culture bag was controlled to 30%. After about 2 hours, the cells sank and the oxygen concentration became stable. The oxygen concentration at that time was 5%.
In this case, the oxygen permeability G can be calculated as follows.
G = Cell filling amount per unit area x Oxygen consumption per cell / Change in oxygen concentration = 1.9e6 x 1.74e-9 / (30 / 100-5 / 100)
= 1.32e-2 (mg / (cm 2・ hr ・ atm))
以上の結果から、上記の条件で使用する培養バッグの酸素透過度Gを以下のように決定した。
G=(1.40e-2+1.43e-2+1.47e-2+1.32e-2)/4
=1.4e-2(mg/(cm2・hr・atm)) From the above results, the oxygen permeability G of the culture bag used under the above conditions was determined as follows.
G = (1.40e-2 + 1.43e-2 + 1.47e-2 + 1.32e-2) / 4
= 1.4e-2 (mg / (cm 2・ hr ・ atm))
G=(1.40e-2+1.43e-2+1.47e-2+1.32e-2)/4
=1.4e-2(mg/(cm2・hr・atm)) From the above results, the oxygen permeability G of the culture bag used under the above conditions was determined as follows.
G = (1.40e-2 + 1.43e-2 + 1.47e-2 + 1.32e-2) / 4
= 1.4e-2 (mg / (cm 2・ hr ・ atm))
細胞の1個当たりの酸素消費量(mg/(hr・個))は、以下のように算出することができる。
まず、ガス透過しない密閉容器に予め細胞数を把握した状態で細胞と培地を注入する。密閉容器に極力気泡が混入していない状態になるようにシリンジ等を用いて気泡を排出する。密閉容器の容積から溶存酸素量を算出する。 The oxygen consumption per cell (mg / (hr ·)) can be calculated as follows.
First, cells and a medium are injected into a closed container that does not permeate gas with the number of cells grasped in advance. Use a syringe or the like to discharge air bubbles so that air bubbles are not mixed in the closed container as much as possible. Calculate the amount of dissolved oxygen from the volume of the closed container.
まず、ガス透過しない密閉容器に予め細胞数を把握した状態で細胞と培地を注入する。密閉容器に極力気泡が混入していない状態になるようにシリンジ等を用いて気泡を排出する。密閉容器の容積から溶存酸素量を算出する。 The oxygen consumption per cell (mg / (hr ·)) can be calculated as follows.
First, cells and a medium are injected into a closed container that does not permeate gas with the number of cells grasped in advance. Use a syringe or the like to discharge air bubbles so that air bubbles are not mixed in the closed container as much as possible. Calculate the amount of dissolved oxygen from the volume of the closed container.
具体的には、10mlの容積の密閉容器に37℃に加温した培地を充填した。37℃(大気中酸素濃度 約21%)で10mlの培地中には、6.86e-2mgの酸素が溶けている(6.86e-2mg/l@37℃(飽和溶存酸素))。培養中の密閉容器における酸素濃度変化を測定して、1時間あたりの溶存酸素の低下濃度を算出する。
PBMCを単位面積当たりの細胞充填量2e6個/mlの密度で10ml密閉容器に注入した場合、容器内には2e7個の細胞が浮遊する。その際の酸素濃度の低下は、0.1%/分であり、1時間当たりでは6%低下した。 Specifically, a closed container having a volume of 10 ml was filled with a medium heated to 37 ° C. 6.86e-2mg of oxygen is dissolved in 10ml of medium at 37 ° C (atmospheric oxygen concentration of about 21%) (6.86e-2mg / l @ 37 ° C (saturated dissolved oxygen)). The change in oxygen concentration in the closed container during culture is measured, and the decrease concentration of dissolved oxygen per hour is calculated.
When PBMC is injected into a 10 ml closed container with a cell filling amount of 2e6 cells / ml per unit area, 2e7 cells float in the container. The decrease in oxygen concentration at that time was 0.1% / min, and decreased by 6% per hour.
PBMCを単位面積当たりの細胞充填量2e6個/mlの密度で10ml密閉容器に注入した場合、容器内には2e7個の細胞が浮遊する。その際の酸素濃度の低下は、0.1%/分であり、1時間当たりでは6%低下した。 Specifically, a closed container having a volume of 10 ml was filled with a medium heated to 37 ° C. 6.86e-2mg of oxygen is dissolved in 10ml of medium at 37 ° C (atmospheric oxygen concentration of about 21%) (6.86e-2mg / l @ 37 ° C (saturated dissolved oxygen)). The change in oxygen concentration in the closed container during culture is measured, and the decrease concentration of dissolved oxygen per hour is calculated.
When PBMC is injected into a 10 ml closed container with a cell filling amount of 2e6 cells / ml per unit area, 2e7 cells float in the container. The decrease in oxygen concentration at that time was 0.1% / min, and decreased by 6% per hour.
21%の飽和溶存酸素に対し6%低下は、6/21=0.29分の低下にあたるから、単位時間当たりの酸素消費量は、以下のとおりである。
6.86e-2mg×0.29=1.99e-2mg/hr
よって、PBMCの細胞の1個当たりの酸素消費量S(mg/(hr・個))は、以下のように算出された。
S=1.99e-2/2e7=1.0e-9mg/(hr・個) A 6% decrease with respect to 21% saturated dissolved oxygen corresponds to a decrease of 6/21 = 0.29 minutes, so the oxygen consumption per unit time is as follows.
6.86e-2mg x 0.29 = 1.99e-2mg / hr
Therefore, the oxygen consumption S (mg / (hr ·) per cell of PBMC was calculated as follows.
S = 1.99e-2 / 2e7 = 1.0e-9mg / (hr / piece)
6.86e-2mg×0.29=1.99e-2mg/hr
よって、PBMCの細胞の1個当たりの酸素消費量S(mg/(hr・個))は、以下のように算出された。
S=1.99e-2/2e7=1.0e-9mg/(hr・個) A 6% decrease with respect to 21% saturated dissolved oxygen corresponds to a decrease of 6/21 = 0.29 minutes, so the oxygen consumption per unit time is as follows.
6.86e-2mg x 0.29 = 1.99e-2mg / hr
Therefore, the oxygen consumption S (mg / (hr ·) per cell of PBMC was calculated as follows.
S = 1.99e-2 / 2e7 = 1.0e-9mg / (hr / piece)
同様の方法で、Jurkatの細胞1個の酸素消費量S(mg/(hr・個))は、以下のように算出された。
S=1.74e-9mg/(hr・個) In the same way, the oxygen consumption S (mg / (hr ·)) of one Jurkat cell was calculated as follows.
S = 1.74e-9mg / (hr / piece)
S=1.74e-9mg/(hr・個) In the same way, the oxygen consumption S (mg / (hr ·)) of one Jurkat cell was calculated as follows.
S = 1.74e-9mg / (hr / piece)
酸素濃度センサの測定値は、蛍光式酸素濃度センサ21によって計測された培養容器20内における培養面付近の酸素濃度であり、蛍光受発光部22から蛍光式センサ入出力部62を介して増殖検出部631に入力される。
The measured value of the oxygen concentration sensor is the oxygen concentration near the culture surface in the culture vessel 20 measured by the fluorescent oxygen concentration sensor 21, and the growth is detected from the fluorescent light receiving / receiving unit 22 via the fluorescent sensor input / output unit 62. It is input to the unit 631.
培養容器の周囲の酸素濃度は、培養容器20が収容されたインキュベータ10内の酸素濃度である。インキュベータ10内の酸素濃度を制御していない状態、あるいはインキュベータ10を使用しない場合は、大気中の酸素濃度である(約21%)。これは、制御部63において事前に設定される。
The oxygen concentration around the culture container is the oxygen concentration in the incubator 10 in which the culture container 20 is housed. When the oxygen concentration in the incubator 10 is not controlled, or when the incubator 10 is not used, it is the oxygen concentration in the atmosphere (about 21%). This is preset in the control unit 63.
ここで、静置培養している状態では、培養容器を透過する酸素量と細胞が消費する酸素量が一致してつり合っているため、以下の式が成り立つ。
G×(M/100-D/100)=C×S
G:ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))
M:培養容器の周囲の酸素濃度(%)
D:酸素濃度センサの測定値(%)
C:細胞の単位面積当たりの個数(個/cm2)
S:細胞1個の単位時間当たりの酸素消費量(mg/(hr・個)) Here, in the state of static culture, the amount of oxygen permeating through the culture vessel and the amount of oxygen consumed by the cells are in agreement and balanced, so that the following equation holds.
G × (M / 100-D / 100) = C × S
G: Oxygen permeability of gas permeable member (mg / (cm 2・ hr ・ atm))
M: Oxygen concentration around the culture vessel (%)
D: Measured value (%) of oxygen concentration sensor
C: Number of cells per unit area (pieces / cm 2 )
S: Oxygen consumption per unit time per cell (mg / (hr ·))
G×(M/100-D/100)=C×S
G:ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))
M:培養容器の周囲の酸素濃度(%)
D:酸素濃度センサの測定値(%)
C:細胞の単位面積当たりの個数(個/cm2)
S:細胞1個の単位時間当たりの酸素消費量(mg/(hr・個)) Here, in the state of static culture, the amount of oxygen permeating through the culture vessel and the amount of oxygen consumed by the cells are in agreement and balanced, so that the following equation holds.
G × (M / 100-D / 100) = C × S
G: Oxygen permeability of gas permeable member (mg / (cm 2・ hr ・ atm))
M: Oxygen concentration around the culture vessel (%)
D: Measured value (%) of oxygen concentration sensor
C: Number of cells per unit area (pieces / cm 2 )
S: Oxygen consumption per unit time per cell (mg / (hr ·))
したがって、本実施形態の細胞培養システムにおいて、増殖検出部631は、細胞の増殖性として、細胞の単位面積当たりの個数を、以下の式(1)によって算出することができる。
C=G×(M/100-D/100)/S ・・・式(1)
C:細胞の単位面積当たりの個数(個/cm2)
G:ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))
M:培養容器の周囲の酸素濃度(%)
D:酸素濃度センサの測定値(%)
S:細胞1個の単位時間当たりの酸素消費量(mg/(hr・個)) Therefore, in the cell culture system of the present embodiment, thegrowth detection unit 631 can calculate the number of cells per unit area as the proliferative property of the cells by the following formula (1).
C = G × (M / 100-D / 100) / S ... Expression (1)
C: Number of cells per unit area (pieces / cm 2 )
G: Oxygen permeability of gas permeable member (mg / (cm 2・ hr ・ atm))
M: Oxygen concentration around the culture vessel (%)
D: Measured value (%) of oxygen concentration sensor
S: Oxygen consumption per unit time per cell (mg / (hr ·))
C=G×(M/100-D/100)/S ・・・式(1)
C:細胞の単位面積当たりの個数(個/cm2)
G:ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))
M:培養容器の周囲の酸素濃度(%)
D:酸素濃度センサの測定値(%)
S:細胞1個の単位時間当たりの酸素消費量(mg/(hr・個)) Therefore, in the cell culture system of the present embodiment, the
C = G × (M / 100-D / 100) / S ... Expression (1)
C: Number of cells per unit area (pieces / cm 2 )
G: Oxygen permeability of gas permeable member (mg / (cm 2・ hr ・ atm))
M: Oxygen concentration around the culture vessel (%)
D: Measured value (%) of oxygen concentration sensor
S: Oxygen consumption per unit time per cell (mg / (hr ·))
本実施形態の細胞培養システムによって算出される細胞の単位面積当たりの個数は、実際の細胞数を厳密に計測するものではなく、培養容器20の培養環境にもとづいて、細胞数を概算で推定するものである。
細胞の大量培養にあたっては、培養が適切に行われているかを確認することが重要であるため、培養容器内の細胞数を概算で推定することは非常に有益である。 The number of cells per unit area calculated by the cell culture system of the present embodiment does not strictly measure the actual number of cells, but estimates the number of cells roughly based on the culture environment of theculture vessel 20. It is a thing.
In mass culture of cells, it is important to confirm that the culture is performed properly, so it is very useful to estimate the number of cells in the culture vessel.
細胞の大量培養にあたっては、培養が適切に行われているかを確認することが重要であるため、培養容器内の細胞数を概算で推定することは非常に有益である。 The number of cells per unit area calculated by the cell culture system of the present embodiment does not strictly measure the actual number of cells, but estimates the number of cells roughly based on the culture environment of the
In mass culture of cells, it is important to confirm that the culture is performed properly, so it is very useful to estimate the number of cells in the culture vessel.
また、本実施形態の細胞培養システムにおいて、増殖検出部631が、培養容器20に複数回新培地を追加した時の酸素濃度の上昇ピーク値の変化の傾きと、培養容器20の安定時の酸素濃度の変化の傾きとの比率にもとづいて得られる所望の時点の培養容器20内の酸素濃度の推定値を、上記式(1)の酸素濃度センサの測定値Dとして用いるようにすることも好ましい。
Further, in the cell culture system of the present embodiment, the growth detection unit 631 shows the gradient of the change in the peak value of the increase in oxygen concentration when the new medium is added to the culture vessel 20 multiple times, and the oxygen when the culture vessel 20 is stable. It is also preferable to use the estimated value of the oxygen concentration in the culture vessel 20 at a desired time point obtained based on the ratio with the gradient of the change in concentration as the measured value D of the oxygen concentration sensor of the above formula (1). ..
すなわち、後述する試験5に示すとおり、細胞培養が進行して培養容器20内の細胞密度が高密度になると、培養容器20の周囲の酸素濃度を高濃度にしても、培養容器20内における培養面付近の酸素濃度が1%未満になり、蛍光式酸素濃度センサ21による測定限界を超えて正確に測定できなくなる。
本実施形態の細胞培養システムによれば、このような場合においても、上記の方法によって、培養容器20内の細胞の単位面積当たりの個数を概算で推定することが可能である。 That is, as shown inTest 5 described later, when cell culture progresses and the cell density in the culture vessel 20 becomes high, even if the oxygen concentration around the culture vessel 20 is high, the culture in the culture vessel 20 is performed. The oxygen concentration near the surface becomes less than 1%, and the measurement limit by the fluorescent oxygen concentration sensor 21 is exceeded, making accurate measurement impossible.
According to the cell culture system of the present embodiment, even in such a case, it is possible to roughly estimate the number of cells per unit area in theculture vessel 20 by the above method.
本実施形態の細胞培養システムによれば、このような場合においても、上記の方法によって、培養容器20内の細胞の単位面積当たりの個数を概算で推定することが可能である。 That is, as shown in
According to the cell culture system of the present embodiment, even in such a case, it is possible to roughly estimate the number of cells per unit area in the
すなわち、培養バッグに複数回新培地を追加した時の酸素濃度の上昇ピーク値の変化の傾きと培養バッグの安定時の酸素濃度の変化の傾きとの比率にもとづいて得られる所望の時点の培養バッグ内の酸素濃度の推定値D’を、酸素濃度センサの測定値として用いることで、以下の式(2)にもとづき細胞の単位面積当たりの個数を計算することができる。
C=G×(M/100-D’/100)/S ・・・式(2) That is, the culture at a desired time point obtained based on the ratio of the slope of the change in the peak value of the increase in oxygen concentration when the new medium was added to the culture bag multiple times and the slope of the change in the oxygen concentration when the culture bag was stable. By using the estimated value D'of the oxygen concentration in the bag as the measured value of the oxygen concentration sensor, the number of cells per unit area can be calculated based on the following formula (2).
C = G × (M / 100-D'/ 100) / S ... Expression (2)
C=G×(M/100-D’/100)/S ・・・式(2) That is, the culture at a desired time point obtained based on the ratio of the slope of the change in the peak value of the increase in oxygen concentration when the new medium was added to the culture bag multiple times and the slope of the change in the oxygen concentration when the culture bag was stable. By using the estimated value D'of the oxygen concentration in the bag as the measured value of the oxygen concentration sensor, the number of cells per unit area can be calculated based on the following formula (2).
C = G × (M / 100-D'/ 100) / S ... Expression (2)
なお、新培地を追加した時の酸素濃度の上昇ピーク時の酸素濃度は、追加する培地の温度に影響を受ける。このため、培地の温度を一定に保持して追加することが好ましい。
また、推定値D’の算出方法については、試験5において詳述する。 The rise in oxygen concentration when a new medium is added The oxygen concentration at the peak is affected by the temperature of the medium to be added. Therefore, it is preferable to keep the temperature of the medium constant and add the medium.
Further, the calculation method of the estimated value D'will be described in detail inTest 5.
また、推定値D’の算出方法については、試験5において詳述する。 The rise in oxygen concentration when a new medium is added The oxygen concentration at the peak is affected by the temperature of the medium to be added. Therefore, it is preferable to keep the temperature of the medium constant and add the medium.
Further, the calculation method of the estimated value D'will be described in detail in
操作部64は、タッチパネルなどの表示部を備え、ユーザによる入力情報を制御部63に送信してPLCの設定などを実行する。また、制御部63からの入力情報を表示する。
電源部65(安定化電源など)は、制御装置60内の各部に電気を供給する。
また、図示しないが、制御装置60において、さらに継電部(リレー)や配線遮断部(ブレーカー)を備えた構成とすることができる。また、制御装置60における制御部63や操作部64などの一部又は全部の構成をマイコンやコンピュータにより実現してもよい。 Theoperation unit 64 includes a display unit such as a touch panel, transmits input information by the user to the control unit 63, and executes PLC setting and the like. In addition, the input information from the control unit 63 is displayed.
The power supply unit 65 (stabilized power supply or the like) supplies electricity to each unit in thecontrol device 60.
Further, although not shown, thecontrol device 60 may be further provided with a relay and a molded case circuit breaker. Further, a part or all of the configuration of the control unit 63, the operation unit 64, etc. in the control device 60 may be realized by a microcomputer or a computer.
電源部65(安定化電源など)は、制御装置60内の各部に電気を供給する。
また、図示しないが、制御装置60において、さらに継電部(リレー)や配線遮断部(ブレーカー)を備えた構成とすることができる。また、制御装置60における制御部63や操作部64などの一部又は全部の構成をマイコンやコンピュータにより実現してもよい。 The
The power supply unit 65 (stabilized power supply or the like) supplies electricity to each unit in the
Further, although not shown, the
本実施形態の細胞の増殖性の検出方法は、少なくとも一部がガス透過性部材からなる培養容器によって細胞を静置状態で培養する細胞培養における細胞の増殖性の検出方法であって、培養容器内における培養面に配置された酸素濃度センサにより培養容器内における培養面付近の酸素濃度を計測し、ガス透過性部材の酸素透過度、酸素濃度センサの測定値、培養容器の周囲の酸素濃度、及び細胞の1個当たりの酸素消費量にもとづき細胞の単位面積当たりの個数を計算することを特徴とする。
The cell proliferative detection method of the present embodiment is a cell proliferative detection method in cell culture in which cells are cultured in a stationary state by a culture vessel having at least a part of a gas permeable member, and is a culture vessel. The oxygen concentration near the culture surface in the culture vessel is measured by the oxygen concentration sensor placed on the culture surface inside, and the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, It is characterized by calculating the number of cells per unit area based on the amount of oxygen consumed per cell.
本実施形態の細胞の増殖性の検出方法において、細胞の単位面積当たりの個数は、以下の式(1)によって算出することができる。
C=G×(M/100-D/100)/S ・・・式(1)
C:細胞の単位面積当たりの個数(個/cm2)
G:ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))
M:培養容器の周囲の酸素濃度(%)
D:酸素濃度センサの測定値(%)
S:細胞1個の単位時間当たりの酸素消費量(mg/(hr・個) ) In the method for detecting cell proliferation of the present embodiment, the number of cells per unit area can be calculated by the following formula (1).
C = G × (M / 100-D / 100) / S ... Expression (1)
C: Number of cells per unit area (pieces / cm 2 )
G: Oxygen permeability of gas permeable member (mg / (cm 2・ hr ・ atm))
M: Oxygen concentration around the culture vessel (%)
D: Measured value (%) of oxygen concentration sensor
S: Oxygen consumption per unit time per cell (mg / (hr ·))
C=G×(M/100-D/100)/S ・・・式(1)
C:細胞の単位面積当たりの個数(個/cm2)
G:ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))
M:培養容器の周囲の酸素濃度(%)
D:酸素濃度センサの測定値(%)
S:細胞1個の単位時間当たりの酸素消費量(mg/(hr・個) ) In the method for detecting cell proliferation of the present embodiment, the number of cells per unit area can be calculated by the following formula (1).
C = G × (M / 100-D / 100) / S ... Expression (1)
C: Number of cells per unit area (pieces / cm 2 )
G: Oxygen permeability of gas permeable member (mg / (cm 2・ hr ・ atm))
M: Oxygen concentration around the culture vessel (%)
D: Measured value (%) of oxygen concentration sensor
S: Oxygen consumption per unit time per cell (mg / (hr ·))
また、本実施形態の細胞の増殖性の検出方法において、培養容器20に複数回新培地を追加した時の酸素濃度の上昇ピーク値の変化の傾きと、培養容器20の安定時の酸素濃度の変化の傾きとの比率にもとづいて得られる所望の時点の培養容器20内の酸素濃度の推定値を、上記式(1)の酸素濃度センサの測定値Dとして用いるようにすることも好ましい。
Further, in the method for detecting cell proliferation of the present embodiment, the slope of the change in the peak value of the increase in oxygen concentration when the new medium is added to the culture vessel 20 multiple times, and the oxygen concentration when the culture vessel 20 is stable. It is also preferable to use the estimated value of the oxygen concentration in the culture vessel 20 at a desired time point obtained based on the ratio with the slope of the change as the measured value D of the oxygen concentration sensor of the above formula (1).
以上のとおり、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法によれば、ガス透過性部材からなる培養容器によって細胞を静置状態で培養する場合において、培養中の所望のタイミングにおける培養容器内の細胞の増殖性を検出することができ、細胞の増殖性として、細胞の単位面積当たりの個数を計算することができる。したがって、本実施形態によれば、細胞の大量培養にあたり、培養が適切に行われているかを確認することが可能となる。
As described above, according to the cell culture system of the present embodiment and the method for detecting cell proliferation, when cells are cultured in a static state in a culture vessel made of a gas permeable member, the desired timing during the culture is achieved. The proliferation of cells in the culture vessel can be detected, and the number of cells per unit area can be calculated as the proliferation of cells. Therefore, according to the present embodiment, it is possible to confirm whether or not the culture is properly performed in the mass culture of cells.
以下、本発明の実施形態に係る細胞培養システム、及び細胞の増殖性の検出方法の効果を確認するために行った試験について説明する。
試験1ではJurkat(リンパ球株化細胞)を培養して増殖性の検出を行った。この試験では、試験期間において培養を中断することなく連続して培養し、細胞密度の推定値と、本実施形態による細胞の単位面積当たりの個数の計算値との比較を行った。
試験2ではPBMC(Peripheral Blood Mononuclear Cells,末梢血単核細胞)を培養して、培養後の細胞密度の実測値と、本実施形態による細胞の単位面積当たりの個数の計算値との比較を行った。 Hereinafter, a test conducted to confirm the effect of the cell culture system according to the embodiment of the present invention and the method for detecting cell proliferation will be described.
In Test 1, Jurkat (lymphocyte cell line) was cultured to detect proliferativeness. In this test, the cells were continuously cultured without interruption during the test period, and the estimated value of the cell density was compared with the calculated value of the number of cells per unit area according to the present embodiment.
InTest 2, PBMC (Peripheral Blood Mononuclear Cells) was cultured, and the measured value of the cell density after the culture was compared with the calculated value of the number of cells per unit area according to the present embodiment. rice field.
試験1ではJurkat(リンパ球株化細胞)を培養して増殖性の検出を行った。この試験では、試験期間において培養を中断することなく連続して培養し、細胞密度の推定値と、本実施形態による細胞の単位面積当たりの個数の計算値との比較を行った。
試験2ではPBMC(Peripheral Blood Mononuclear Cells,末梢血単核細胞)を培養して、培養後の細胞密度の実測値と、本実施形態による細胞の単位面積当たりの個数の計算値との比較を行った。 Hereinafter, a test conducted to confirm the effect of the cell culture system according to the embodiment of the present invention and the method for detecting cell proliferation will be described.
In Test 1, Jurkat (lymphocyte cell line) was cultured to detect proliferativeness. In this test, the cells were continuously cultured without interruption during the test period, and the estimated value of the cell density was compared with the calculated value of the number of cells per unit area according to the present embodiment.
In
試験3ではPBMCを培養すると共に、所定のタイミングで培養を中断して実際の細胞数をカウントし、得られた細胞密度の実測値と本実施形態による細胞の単位面積当たりの個数の計算値との比較を行った。
試験4ではiPS細胞を培養して、培養終了後に実際の細胞数をカウントし、得られた細胞密度の実測値と本実施形態による細胞の単位面積当たりの個数の計算値との比較を行った。 In Test 3, PBMC was cultured, the culture was interrupted at a predetermined timing, the actual number of cells was counted, the measured value of the obtained cell density and the calculated value of the number of cells per unit area according to the present embodiment. Was compared.
InTest 4, iPS cells were cultured, the actual number of cells was counted after the culture was completed, and the measured value of the obtained cell density was compared with the calculated value of the number of cells per unit area according to the present embodiment. ..
試験4ではiPS細胞を培養して、培養終了後に実際の細胞数をカウントし、得られた細胞密度の実測値と本実施形態による細胞の単位面積当たりの個数の計算値との比較を行った。 In Test 3, PBMC was cultured, the culture was interrupted at a predetermined timing, the actual number of cells was counted, the measured value of the obtained cell density and the calculated value of the number of cells per unit area according to the present embodiment. Was compared.
In
試験5は、試験3による培養の続きの期間についてのものであり、培養容器内の細胞密度が高密度となって、培養面付近の酸素濃度の測定値が1%未満の期間を含むものである。培養終了後に実際の細胞数をカウントし、得られた細胞密度の実測値と本実施形態による細胞の単位面積当たりの個数の計算値との比較を行った。
Test 5 is for the continuation period of the culture by the test 3, and includes the period in which the cell density in the culture vessel becomes high and the measured value of the oxygen concentration near the culture surface is less than 1%. After the culture was completed, the actual number of cells was counted, and the measured value of the obtained cell density was compared with the calculated value of the number of cells per unit area according to the present embodiment.
[試験1]
培養バッグとして、厚み110μmの直鎖状低密度ポリエチレンからなり、外形のサイズが120mm×65mm、培養空間の底面積が48cm2である培養バッグ(東洋製罐グループホールディングス株式会社製)を準備した。この培養バッグは、2枚のフラットなフィルムの周縁部をヒートシールすることにより形成した。この培養バッグにおける酸素透過度は、1.4e-2(mg/(cm2・hr・atm))であった。 [Test 1]
As a culture bag, a culture bag (manufactured by Toyo Seikan Group Holdings Co., Ltd.) made of linear low-density polyethylene having a thickness of 110 μm, having an outer size of 120 mm × 65 mm and a bottom area of the culture space of 48 cm 2 was prepared. The culture bag was formed by heat-sealing the edges of two flat films. The oxygen permeability in this culture bag was 1.4e-2 (mg / (cm 2 · hr · atm)).
培養バッグとして、厚み110μmの直鎖状低密度ポリエチレンからなり、外形のサイズが120mm×65mm、培養空間の底面積が48cm2である培養バッグ(東洋製罐グループホールディングス株式会社製)を準備した。この培養バッグは、2枚のフラットなフィルムの周縁部をヒートシールすることにより形成した。この培養バッグにおける酸素透過度は、1.4e-2(mg/(cm2・hr・atm))であった。 [Test 1]
As a culture bag, a culture bag (manufactured by Toyo Seikan Group Holdings Co., Ltd.) made of linear low-density polyethylene having a thickness of 110 μm, having an outer size of 120 mm × 65 mm and a bottom area of the culture space of 48 cm 2 was prepared. The culture bag was formed by heat-sealing the edges of two flat films. The oxygen permeability in this culture bag was 1.4e-2 (mg / (cm 2 · hr · atm)).
この培養バッグ内面のほぼ中央に蛍光式酸素濃度センサ(センサチップPSt3タイプ,PreSens社)を固定した。
この培養バッグを押圧治具に挟み込み加圧した状態で培養を行った。押圧治具の底面は透明の樹脂板であり、樹脂板における蛍光式酸素濃度センサに対応する位置に蛍光受発光部を固定した。蛍光受発光部は、制御装置における蛍光式センサ入出力部に付属し、蛍光式センサ入出力部としては、OXY-4mini(タイテック株式会社)を使用した。
使用した蛍光式酸素濃度センサ、蛍光受発光部、及び蛍光式センサ入出力部については、以下の試験においても同様である。 A fluorescent oxygen concentration sensor (sensor chip PSt3 type, PreSens) was fixed in the center of the inner surface of the culture bag.
This culture bag was sandwiched between pressing jigs and cultured under pressure. The bottom surface of the pressing jig is a transparent resin plate, and the fluorescence receiving / emitting unit is fixed at a position corresponding to the fluorescent oxygen concentration sensor on the resin plate. The fluorescence receiving / emitting unit was attached to the fluorescence sensor input / output unit in the control device, and OXY-4mini (Titec Co., Ltd.) was used as the fluorescence sensor input / output unit.
The same applies to the fluorescent oxygen concentration sensor, the fluorescent light receiving / emitting unit, and the fluorescent sensor input / output unit used in the following tests.
この培養バッグを押圧治具に挟み込み加圧した状態で培養を行った。押圧治具の底面は透明の樹脂板であり、樹脂板における蛍光式酸素濃度センサに対応する位置に蛍光受発光部を固定した。蛍光受発光部は、制御装置における蛍光式センサ入出力部に付属し、蛍光式センサ入出力部としては、OXY-4mini(タイテック株式会社)を使用した。
使用した蛍光式酸素濃度センサ、蛍光受発光部、及び蛍光式センサ入出力部については、以下の試験においても同様である。 A fluorescent oxygen concentration sensor (sensor chip PSt3 type, PreSens) was fixed in the center of the inner surface of the culture bag.
This culture bag was sandwiched between pressing jigs and cultured under pressure. The bottom surface of the pressing jig is a transparent resin plate, and the fluorescence receiving / emitting unit is fixed at a position corresponding to the fluorescent oxygen concentration sensor on the resin plate. The fluorescence receiving / emitting unit was attached to the fluorescence sensor input / output unit in the control device, and OXY-4mini (Titec Co., Ltd.) was used as the fluorescence sensor input / output unit.
The same applies to the fluorescent oxygen concentration sensor, the fluorescent light receiving / emitting unit, and the fluorescent sensor input / output unit used in the following tests.
細胞としては、Jurkat(ヒト白血病T細胞由来の細胞株)を1.20e6個/cm2の細胞密度(細胞の単位面積当たりの個数)で播種した。
培地としては、AlyS505N-0(株式会社細胞科学研究所製)にFBS2%を添加したものを使用し、培養バッグ内に35mlの培地を細胞と共に充填した。 As cells, Jurkat (cell line derived from human leukemia T cells) was seeded at a cell density of 1.20 e6 cells / cm 2 (number per unit area of cells).
As the medium, AlyS505N-0 (manufactured by Cell Science Laboratory Co., Ltd.) to whichFBS 2% was added was used, and 35 ml of the medium was filled in the culture bag together with the cells.
培地としては、AlyS505N-0(株式会社細胞科学研究所製)にFBS2%を添加したものを使用し、培養バッグ内に35mlの培地を細胞と共に充填した。 As cells, Jurkat (cell line derived from human leukemia T cells) was seeded at a cell density of 1.20 e6 cells / cm 2 (number per unit area of cells).
As the medium, AlyS505N-0 (manufactured by Cell Science Laboratory Co., Ltd.) to which
インキュベータとしては、高酸素培養可能なマルチガスインキュベータ(PHC株式会社,型式MCO-5M-PJ)を使用し、インキュベータ内に自動送液機構を設置した。
培地を充填した培地供給バッグを10℃の保冷庫に保管すると共に、培養バッグにチューブで接続した。また、廃液バッグを培養バッグにチューブで接続した。培地の送液は、チューブポンプの制御により行った。培地は、37℃のインキュベータ内を通過する間に加温されて、培養バッグに入る時点では約30℃になった。
チューブポンプの制御及びインキュベータの酸素濃度の制御は、制御装置における制御部により行った。なお、各試験において、制御部における増殖検出部は、コンピュータにより構成した。 As an incubator, a multi-gas incubator capable of high oxygen culture (PHC Corporation, model MCO-5M-PJ) was used, and an automatic liquid feeding mechanism was installed in the incubator.
The medium supply bag filled with the medium was stored in a cold storage at 10 ° C. and connected to the culture bag with a tube. In addition, the waste liquid bag was connected to the culture bag with a tube. The medium was fed under the control of a tube pump. The medium was warmed while passing through the 37 ° C incubator to about 30 ° C by the time it entered the culture bag.
The control of the tube pump and the control of the oxygen concentration of the incubator were performed by the control unit in the control device. In each test, the proliferation detection unit in the control unit was configured by a computer.
培地を充填した培地供給バッグを10℃の保冷庫に保管すると共に、培養バッグにチューブで接続した。また、廃液バッグを培養バッグにチューブで接続した。培地の送液は、チューブポンプの制御により行った。培地は、37℃のインキュベータ内を通過する間に加温されて、培養バッグに入る時点では約30℃になった。
チューブポンプの制御及びインキュベータの酸素濃度の制御は、制御装置における制御部により行った。なお、各試験において、制御部における増殖検出部は、コンピュータにより構成した。 As an incubator, a multi-gas incubator capable of high oxygen culture (PHC Corporation, model MCO-5M-PJ) was used, and an automatic liquid feeding mechanism was installed in the incubator.
The medium supply bag filled with the medium was stored in a cold storage at 10 ° C. and connected to the culture bag with a tube. In addition, the waste liquid bag was connected to the culture bag with a tube. The medium was fed under the control of a tube pump. The medium was warmed while passing through the 37 ° C incubator to about 30 ° C by the time it entered the culture bag.
The control of the tube pump and the control of the oxygen concentration of the incubator were performed by the control unit in the control device. In each test, the proliferation detection unit in the control unit was configured by a computer.
そして、指定の時間に自動で培地の一部交換を実施することで、培養によって劣化した培地を培地バッグから排出し、新しい培地を充填した。
具体的には、培養開始から所定のタイミングで、培地交換を実施した。このとき、培地排出量は、35ml中25mlとした。送液速度は2ml/分とした。培地交換によって新培地が追加されると、培養バッグ内の酸素濃度が一時的に上昇し、後述するグラフにおいてピークが表示された。 Then, by automatically exchanging a part of the medium at a designated time, the medium deteriorated by the culture was discharged from the medium bag and filled with a new medium.
Specifically, the medium was exchanged at a predetermined timing from the start of the culture. At this time, the amount of medium discharged was 25 ml out of 35 ml. The liquid feeding rate was 2 ml / min. When new medium was added by medium exchange, the oxygen concentration in the culture bag temporarily increased, and a peak was displayed in the graph described later.
具体的には、培養開始から所定のタイミングで、培地交換を実施した。このとき、培地排出量は、35ml中25mlとした。送液速度は2ml/分とした。培地交換によって新培地が追加されると、培養バッグ内の酸素濃度が一時的に上昇し、後述するグラフにおいてピークが表示された。 Then, by automatically exchanging a part of the medium at a designated time, the medium deteriorated by the culture was discharged from the medium bag and filled with a new medium.
Specifically, the medium was exchanged at a predetermined timing from the start of the culture. At this time, the amount of medium discharged was 25 ml out of 35 ml. The liquid feeding rate was 2 ml / min. When new medium was added by medium exchange, the oxygen concentration in the culture bag temporarily increased, and a peak was displayed in the graph described later.
また、インキュベータの酸素濃度を指定のタイミングで増加させた。
具体的には、培養開始から23時間後のタイミングで、21%から25%に変更した。また、培養開始から32時間後のタイミングで、25%から30%に変更した。また、培養開始から50時間後のタイミングで、30%から35%に変更した。また、培養開始から53時間後のタイミングで、35%から40%に変更した。 In addition, the oxygen concentration in the incubator was increased at the specified timing.
Specifically, it was changed from 21% to 25% at the timing 23 hours after the start of culture. In addition, it was changed from 25% to 30% at the timing 32 hours after the start of culture. In addition, it was changed from 30% to 35% at thetiming 50 hours after the start of culture. In addition, it was changed from 35% to 40% at the timing 53 hours after the start of culture.
具体的には、培養開始から23時間後のタイミングで、21%から25%に変更した。また、培養開始から32時間後のタイミングで、25%から30%に変更した。また、培養開始から50時間後のタイミングで、30%から35%に変更した。また、培養開始から53時間後のタイミングで、35%から40%に変更した。 In addition, the oxygen concentration in the incubator was increased at the specified timing.
Specifically, it was changed from 21% to 25% at the timing 23 hours after the start of culture. In addition, it was changed from 25% to 30% at the timing 32 hours after the start of culture. In addition, it was changed from 30% to 35% at the
試験1の細胞培養における、培養バッグに固定された蛍光式酸素濃度センサによる酸素濃度の測定結果を表すグラフを図4に示す。
この測定結果にもとづいて、細胞密度の推定値と、本実施形態による細胞の単位面積当たりの個数の計算値との比較を、培養開始から10,40,60時間後について行った。 FIG. 4 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of Test 1.
Based on this measurement result, the estimated value of the cell density and the calculated value of the number of cells per unit area according to this embodiment were compared with each other 10, 40, and 60 hours after the start of the culture.
この測定結果にもとづいて、細胞密度の推定値と、本実施形態による細胞の単位面積当たりの個数の計算値との比較を、培養開始から10,40,60時間後について行った。 FIG. 4 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of Test 1.
Based on this measurement result, the estimated value of the cell density and the calculated value of the number of cells per unit area according to this embodiment were compared with each other 10, 40, and 60 hours after the start of the culture.
具体的には、Jurkat細胞の増殖速度は、約1.4倍/24hrであり、比較的安定している。そこで、播種時細胞密度(1.20e6個/cm2)にもとづいて、培養開始から24, 48時間後の細胞密度をそれぞれ1.68e6個/cm2(=1.20e6×1.4)、2.35e6個/cm2(=1.68e6×1.4)と推定し、これらにもとづき10,40,60時間後の細胞密度の推定値を以下のように算出した。
Specifically, the proliferation rate of Jurkat cells is about 1.4 times / 24 hr, which is relatively stable. Therefore, based on the cell density at the time of seeding (1.20e6 cells / cm 2 ), the cell densities 24 and 48 hours after the start of culture were 1.68e6 cells / cm 2 (= 1.20e6 × 1.4) and 2.35e6 cells / cm, respectively. It was estimated to be 2 (= 1.68e6 × 1.4), and based on these, the estimated cell density after 10, 40, and 60 hours was calculated as follows.
10時間後の細胞密度の推定値1.40e6個/cm2(=(1.20e6×1.4-1.20e6)×10/24+1.20e6)
40時間後の細胞密度の推定値2.13e6個/cm2(=(1.68e6×1.4-1.68e6)×16/24+1.68e6)
60時間後の細胞密度の推定値2.82e6個/cm2(=(2.35e6×1.4-2.35e6)×12/24+2.35e6) Estimated cell density after 10 hours 1.40e6 cells / cm 2 (= (1.20e6 × 1.4-1.20e6) × 10/24 + 1.20e6)
Estimated cell density after 40 hours 2.13e6 cells / cm 2 (= (1.68e6 × 1.4-1.68e6) × 16/24 + 1.68e6)
Estimated cell density after 60 hours 2.82e6 cells / cm 2 (= (2.35e6 × 1.4-2.35e6) × 12/24 + 2.35e6)
40時間後の細胞密度の推定値2.13e6個/cm2(=(1.68e6×1.4-1.68e6)×16/24+1.68e6)
60時間後の細胞密度の推定値2.82e6個/cm2(=(2.35e6×1.4-2.35e6)×12/24+2.35e6) Estimated cell density after 10 hours 1.40e6 cells / cm 2 (= (1.20e6 × 1.4-1.20e6) × 10/24 + 1.20e6)
Estimated cell density after 40 hours 2.13e6 cells / cm 2 (= (1.68e6 × 1.4-1.68e6) × 16/24 + 1.68e6)
Estimated cell density after 60 hours 2.82e6 cells / cm 2 (= (2.35e6 × 1.4-2.35e6) × 12/24 + 2.35e6)
また、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法による細胞の単位面積当たりの個数の計算値は、10,40,60時間後について、それぞれ以下のように算出される。まず、ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))は、1.4e-2であった。また、細胞1個の単位時間当たりの酸素消費量(mg/(hr・個))は、1.74e-9であった。
In addition, the calculated values of the number of cells per unit area by the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells are calculated as follows after 10, 40, and 60 hours, respectively. First, the oxygen permeability (mg / (cm 2 · hr · atm)) of the gas permeable member was 1.4e-2. The oxygen consumption per unit time (mg / (hr ·)) of one cell was 1.74e-9.
また、10時間後において、培養容器の周囲の酸素濃度(%)は21%、酸素濃度センサの測定値(%)は、5%であった。
したがって、10時間後の細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(21/100-5/100)/1.74e-9=1.29e6
これに対して、10時間後の細胞密度の推定値は、上述のとおり、1.40e6個/cm2であった。 After 10 hours, the oxygen concentration (%) around the culture vessel was 21%, and the measured value (%) of the oxygen concentration sensor was 5%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells after 10 hours is as follows.
C = 1.4e-2 × (21 / 100-5 / 100) /1.74e-9=1.29e6
On the other hand, the estimated value of the cell density after 10 hours was 1.40e6 cells / cm 2 as described above.
したがって、10時間後の細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(21/100-5/100)/1.74e-9=1.29e6
これに対して、10時間後の細胞密度の推定値は、上述のとおり、1.40e6個/cm2であった。 After 10 hours, the oxygen concentration (%) around the culture vessel was 21%, and the measured value (%) of the oxygen concentration sensor was 5%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells after 10 hours is as follows.
C = 1.4e-2 × (21 / 100-5 / 100) /1.74e-9=1.29e6
On the other hand, the estimated value of the cell density after 10 hours was 1.40e6 cells / cm 2 as described above.
また、40時間後において、培養容器の周囲の酸素濃度(%)は30%、酸素濃度センサの測定値(%)は、3%であった。
したがって、40時間後の細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(30/100-3/100)/1.74e-9=2.17e6
これに対して、40時間後の細胞密度の推定値は、上述のとおり、2.13e6個/cm2であった。 After 40 hours, the oxygen concentration (%) around the culture vessel was 30%, and the measured value (%) of the oxygen concentration sensor was 3%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells after 40 hours is as follows.
C = 1.4e-2 × (30 / 100-3 / 100) /1.74e-9=2.17e6
On the other hand, the estimated value of the cell density after 40 hours was 2.13e6 cells / cm 2 as described above.
したがって、40時間後の細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(30/100-3/100)/1.74e-9=2.17e6
これに対して、40時間後の細胞密度の推定値は、上述のとおり、2.13e6個/cm2であった。 After 40 hours, the oxygen concentration (%) around the culture vessel was 30%, and the measured value (%) of the oxygen concentration sensor was 3%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells after 40 hours is as follows.
C = 1.4e-2 × (30 / 100-3 / 100) /1.74e-9=2.17e6
On the other hand, the estimated value of the cell density after 40 hours was 2.13e6 cells / cm 2 as described above.
さらに、60時間後において、培養容器の周囲の酸素濃度(%)は40%、酸素濃度センサの測定値(%)は、2%であった。
したがって、60時間後の細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(40/100-2/100)/1.74e-9=3.06e6
これに対して、60時間後の細胞密度の推定値は、上述のとおり、2.82e6個/cm2であった。 Furthermore, after 60 hours, the oxygen concentration (%) around the culture vessel was 40%, and the measured value (%) of the oxygen concentration sensor was 2%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells after 60 hours is as follows.
C = 1.4e-2 × (40 / 100-2 / 100) /1.74e-9=3.06e6
On the other hand, the estimated value of the cell density after 60 hours was 2.82e6 cells / cm 2 as described above.
したがって、60時間後の細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(40/100-2/100)/1.74e-9=3.06e6
これに対して、60時間後の細胞密度の推定値は、上述のとおり、2.82e6個/cm2であった。 Furthermore, after 60 hours, the oxygen concentration (%) around the culture vessel was 40%, and the measured value (%) of the oxygen concentration sensor was 2%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells after 60 hours is as follows.
C = 1.4e-2 × (40 / 100-2 / 100) /1.74e-9=3.06e6
On the other hand, the estimated value of the cell density after 60 hours was 2.82e6 cells / cm 2 as described above.
以上のとおり、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法によれば、ガス透過性部材からなる培養容器によって細胞を静置状態で培養する場合において、培養中の所望のタイミングにおける細胞の単位面積当たりの個数の概算を算出できることが分かった。
As described above, according to the cell culture system of the present embodiment and the method for detecting cell proliferation, when cells are cultured in a static state in a culture vessel made of a gas-permeable member, the desired timing during culture is achieved. It was found that the approximate number of cells per unit area in the cell can be calculated.
[試験2]
培養バッグとして、試験1と同じものを準備した。この培養バッグにおける酸素透過度は、1.4e-2(mg/(cm2・hr・atm))であった。
この培養バッグ内面のほぼ中央に蛍光式酸素濃度センサを固定した。
この培養バッグを押圧治具に挟み込み加圧した状態で培養を行った。押圧治具の底面は透明の樹脂板であり、樹脂板における蛍光式酸素濃度センサに対応する位置に蛍光受発光部を固定した。 [Test 2]
The same culture bag as in Test 1 was prepared. The oxygen permeability in this culture bag was 1.4e-2 (mg / (cm 2 · hr · atm)).
A fluorescent oxygen concentration sensor was fixed in the center of the inner surface of the culture bag.
This culture bag was sandwiched between pressing jigs and cultured under pressure. The bottom surface of the pressing jig is a transparent resin plate, and the fluorescence receiving / emitting unit is fixed at a position corresponding to the fluorescent oxygen concentration sensor on the resin plate.
培養バッグとして、試験1と同じものを準備した。この培養バッグにおける酸素透過度は、1.4e-2(mg/(cm2・hr・atm))であった。
この培養バッグ内面のほぼ中央に蛍光式酸素濃度センサを固定した。
この培養バッグを押圧治具に挟み込み加圧した状態で培養を行った。押圧治具の底面は透明の樹脂板であり、樹脂板における蛍光式酸素濃度センサに対応する位置に蛍光受発光部を固定した。 [Test 2]
The same culture bag as in Test 1 was prepared. The oxygen permeability in this culture bag was 1.4e-2 (mg / (cm 2 · hr · atm)).
A fluorescent oxygen concentration sensor was fixed in the center of the inner surface of the culture bag.
This culture bag was sandwiched between pressing jigs and cultured under pressure. The bottom surface of the pressing jig is a transparent resin plate, and the fluorescence receiving / emitting unit is fixed at a position corresponding to the fluorescent oxygen concentration sensor on the resin plate.
細胞としては、PBMC(Peripheral Blood Mononuclear Cells,末梢血単核細胞)を使用した。培養バッグへの播種に先立って、抗CD3抗体を塗工、固層化したフラスコを用いて、PBMCを3日間活性化した後、培養バッグに1.6e6個/cm2の細胞密度で播種した。
As cells, PBMC (Peripheral Blood Mononuclear Cells) was used. Prior to seeding in the culture bag, PBMC was activated for 3 days using a flask coated with anti-CD3 antibody and solidified, and then seeded in the culture bag at a cell density of 1.6 e6 cells / cm 2 .
培地としては、AlyS505N-7(株式会社細胞科学研究所製)にFBS2%を添加したものを使用し、培養バッグ内に35mlの培地を細胞と共に充填した。
培養バッグへの播種後3日間は酸素濃度の測定を行わず、続く3日間の約60hrにおいて培養バッグ内の酸素濃度の測定を行った。 As the medium, AlyS505N-7 (manufactured by Cell Science Laboratory Co., Ltd.) to whichFBS 2% was added was used, and 35 ml of the medium was filled in the culture bag together with the cells.
The oxygen concentration was not measured for 3 days after sowing in the culture bag, and the oxygen concentration in the culture bag was measured for about 60 hours for the following 3 days.
培養バッグへの播種後3日間は酸素濃度の測定を行わず、続く3日間の約60hrにおいて培養バッグ内の酸素濃度の測定を行った。 As the medium, AlyS505N-7 (manufactured by Cell Science Laboratory Co., Ltd.) to which
The oxygen concentration was not measured for 3 days after sowing in the culture bag, and the oxygen concentration in the culture bag was measured for about 60 hours for the following 3 days.
インキュベータとしては、高酸素培養可能なマルチガスインキュベータ(PHC株式会社,型式MCO-5M-PJ)を使用し、インキュベータ内に自動送液機構を設置した。
培地を充填した培地供給バッグを10℃の保冷庫に保管すると共に、培養バッグにチューブで接続した。また、廃液バッグを培養バッグにチューブで接続した。培地の送液は、チューブポンプの制御により行った。 As an incubator, a multi-gas incubator capable of high oxygen culture (PHC Corporation, model MCO-5M-PJ) was used, and an automatic liquid feeding mechanism was installed in the incubator.
The medium supply bag filled with the medium was stored in a cold storage at 10 ° C. and connected to the culture bag with a tube. In addition, the waste liquid bag was connected to the culture bag with a tube. The medium was fed under the control of a tube pump.
培地を充填した培地供給バッグを10℃の保冷庫に保管すると共に、培養バッグにチューブで接続した。また、廃液バッグを培養バッグにチューブで接続した。培地の送液は、チューブポンプの制御により行った。 As an incubator, a multi-gas incubator capable of high oxygen culture (PHC Corporation, model MCO-5M-PJ) was used, and an automatic liquid feeding mechanism was installed in the incubator.
The medium supply bag filled with the medium was stored in a cold storage at 10 ° C. and connected to the culture bag with a tube. In addition, the waste liquid bag was connected to the culture bag with a tube. The medium was fed under the control of a tube pump.
そして、指定の時間に自動で培地の一部交換を実施することで、培養によって劣化した培地を培地バッグから排出し、新しい培地を充填した。
具体的には、培養開始から所定のタイミングで、培地交換を実施した。このとき、培地排出量は、35ml中25mlとした。送液速度は2ml/分とした。培地交換によって新培地が追加されると、培養バッグ内の酸素濃度が一時的に上昇し、後述するグラフにおいてピークが表示された。
また、本試験では、インキュベータ10の酸素濃度を増加させず、高酸素での培養は行わなかった。 Then, by automatically exchanging a part of the medium at a designated time, the medium deteriorated by the culture was discharged from the medium bag and filled with a new medium.
Specifically, the medium was exchanged at a predetermined timing from the start of the culture. At this time, the amount of medium discharged was 25 ml out of 35 ml. The liquid feeding rate was 2 ml / min. When new medium was added by medium exchange, the oxygen concentration in the culture bag temporarily increased, and a peak was displayed in the graph described later.
Moreover, in this test, the oxygen concentration of theincubator 10 was not increased, and the culture in high oxygen was not performed.
具体的には、培養開始から所定のタイミングで、培地交換を実施した。このとき、培地排出量は、35ml中25mlとした。送液速度は2ml/分とした。培地交換によって新培地が追加されると、培養バッグ内の酸素濃度が一時的に上昇し、後述するグラフにおいてピークが表示された。
また、本試験では、インキュベータ10の酸素濃度を増加させず、高酸素での培養は行わなかった。 Then, by automatically exchanging a part of the medium at a designated time, the medium deteriorated by the culture was discharged from the medium bag and filled with a new medium.
Specifically, the medium was exchanged at a predetermined timing from the start of the culture. At this time, the amount of medium discharged was 25 ml out of 35 ml. The liquid feeding rate was 2 ml / min. When new medium was added by medium exchange, the oxygen concentration in the culture bag temporarily increased, and a peak was displayed in the graph described later.
Moreover, in this test, the oxygen concentration of the
そして、測定開始61時間後に培養バッグ内を撹拌して細胞を培地中に均一に浮遊させ、シリンジを用いて培養バッグから培地を約0.5ml採取して、トリパンブルーで染色し血球計数盤を用いて細胞密度を計測した。その結果、測定開始61時間後の細胞密度は、2.34e6個/cm2であった。
試験2の細胞培養における、培養バッグに固定された蛍光式酸素濃度センサによる酸素濃度の測定結果を表すグラフを図5に示す。 Then, 61 hours after the start of measurement, the cells were uniformly suspended in the medium by stirring in the culture bag, about 0.5 ml of the medium was collected from the culture bag using a syringe, stained with tripan blue, and used with a blood cell counter. The cell density was measured. As a result, thecell density 61 hours after the start of measurement was 2.34e6 cells / cm 2 .
FIG. 5 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture ofTest 2.
試験2の細胞培養における、培養バッグに固定された蛍光式酸素濃度センサによる酸素濃度の測定結果を表すグラフを図5に示す。 Then, 61 hours after the start of measurement, the cells were uniformly suspended in the medium by stirring in the culture bag, about 0.5 ml of the medium was collected from the culture bag using a syringe, stained with tripan blue, and used with a blood cell counter. The cell density was measured. As a result, the
FIG. 5 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of
また、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法による細胞の単位面積当たりの個数の計算値は、61時間後について、以下のように算出される。
まず、ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))は、1.4e-2であった。また、細胞1個の単位時間当たりの酸素消費量(mg/(hr・個))は、1.00e-9であった。 Further, the calculated value of the number of cells per unit area by the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells is calculated as follows after 61 hours.
First, the oxygen permeability (mg / (cm 2 · hr · atm)) of the gas permeable member was 1.4e-2. The oxygen consumption per unit time (mg / (hr ·)) of one cell was 1.00e-9.
まず、ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))は、1.4e-2であった。また、細胞1個の単位時間当たりの酸素消費量(mg/(hr・個))は、1.00e-9であった。 Further, the calculated value of the number of cells per unit area by the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells is calculated as follows after 61 hours.
First, the oxygen permeability (mg / (cm 2 · hr · atm)) of the gas permeable member was 1.4e-2. The oxygen consumption per unit time (mg / (hr ·)) of one cell was 1.00e-9.
また、培養容器の周囲の酸素濃度(%)は21%、酸素濃度センサの測定値(%)は、1.2%であった。
したがって、細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(21/100-1.2/100)/1.00e-9=2.77e6
これに対して、細胞密度の実測値は、上述のとおり、2.34e6個/cm2であった。 The oxygen concentration (%) around the culture vessel was 21%, and the measured value (%) of the oxygen concentration sensor was 1.2%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells is as follows.
C = 1.4e-2 × (21 / 100-1.2 / 100) /1.00e-9=2.77e6
On the other hand, the measured value of the cell density was 2.34e6 cells / cm 2 as described above.
したがって、細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(21/100-1.2/100)/1.00e-9=2.77e6
これに対して、細胞密度の実測値は、上述のとおり、2.34e6個/cm2であった。 The oxygen concentration (%) around the culture vessel was 21%, and the measured value (%) of the oxygen concentration sensor was 1.2%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells is as follows.
C = 1.4e-2 × (21 / 100-1.2 / 100) /1.00e-9=2.77e6
On the other hand, the measured value of the cell density was 2.34e6 cells / cm 2 as described above.
すなわち、本試験によっても、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法によれば、ガス透過性部材からなる培養容器によって細胞を静置状態で培養する場合において、細胞の単位面積当たりの個数の概算を算出できることが分かった。
That is, even in this test, according to the cell culture system of the present embodiment and the method for detecting cell proliferation, when cells are cultured in a stationary state in a culture vessel made of a gas permeable member, the cell unit. It was found that an approximate number of cells per area could be calculated.
[試験3]
培養バッグとして、試験1と同じものを準備した。この培養バッグにおける酸素透過度は、1.4e-2(mg/(cm2・hr・atm))であった。
この培養バッグ内面のほぼ中央に蛍光式酸素濃度センサを固定した。
この培養バッグを押圧治具に挟み込み加圧した状態で培養を行った。押圧治具の底面は透明の樹脂板であり、樹脂板における蛍光式酸素濃度センサに対応する位置に蛍光受発光部を固定した。 [Test 3]
The same culture bag as in Test 1 was prepared. The oxygen permeability in this culture bag was 1.4e-2 (mg / (cm 2 · hr · atm)).
A fluorescent oxygen concentration sensor was fixed in the center of the inner surface of the culture bag.
This culture bag was sandwiched between pressing jigs and cultured under pressure. The bottom surface of the pressing jig is a transparent resin plate, and the fluorescence receiving / emitting unit is fixed at a position corresponding to the fluorescent oxygen concentration sensor on the resin plate.
培養バッグとして、試験1と同じものを準備した。この培養バッグにおける酸素透過度は、1.4e-2(mg/(cm2・hr・atm))であった。
この培養バッグ内面のほぼ中央に蛍光式酸素濃度センサを固定した。
この培養バッグを押圧治具に挟み込み加圧した状態で培養を行った。押圧治具の底面は透明の樹脂板であり、樹脂板における蛍光式酸素濃度センサに対応する位置に蛍光受発光部を固定した。 [Test 3]
The same culture bag as in Test 1 was prepared. The oxygen permeability in this culture bag was 1.4e-2 (mg / (cm 2 · hr · atm)).
A fluorescent oxygen concentration sensor was fixed in the center of the inner surface of the culture bag.
This culture bag was sandwiched between pressing jigs and cultured under pressure. The bottom surface of the pressing jig is a transparent resin plate, and the fluorescence receiving / emitting unit is fixed at a position corresponding to the fluorescent oxygen concentration sensor on the resin plate.
細胞としては、PBMC(Peripheral Blood Mononuclear Cells,末梢血単核細胞)を使用した。培養バッグへの播種に先立って、抗CD3抗体を塗工、固層化したフラスコを用いて、PBMCを3日間活性化した後、培養バッグに1.06e6個/cm2の細胞密度で播種した。
As cells, PBMC (Peripheral Blood Mononuclear Cells) was used. Prior to seeding in culture bags, PBMCs were activated for 3 days using a flask coated with anti-CD3 antibody and solidified, and then seeded in culture bags at a cell density of 1.06e6 cells / cm 2 .
培地としては、AlyS505N-7(株式会社細胞科学研究所製)にFBS2%を添加したものを使用し、培養バッグ内に35mlの培地を細胞と共に充填した。
培養バッグへの播種後4日間は酸素濃度の測定を行わず、続く3日間の約65hrにおいて培養バッグ内の酸素濃度の測定を行った。 As the medium, AlyS505N-7 (manufactured by Cell Science Laboratory Co., Ltd.) to whichFBS 2% was added was used, and 35 ml of the medium was filled in the culture bag together with the cells.
The oxygen concentration was not measured for 4 days after sowing in the culture bag, and the oxygen concentration in the culture bag was measured for about 65 hours for the following 3 days.
培養バッグへの播種後4日間は酸素濃度の測定を行わず、続く3日間の約65hrにおいて培養バッグ内の酸素濃度の測定を行った。 As the medium, AlyS505N-7 (manufactured by Cell Science Laboratory Co., Ltd.) to which
The oxygen concentration was not measured for 4 days after sowing in the culture bag, and the oxygen concentration in the culture bag was measured for about 65 hours for the following 3 days.
ただし、試験3では試験2と異なり、細胞数を計測するために、自動送液を停止させて、培養バッグを撹拌して細胞を培地中に均一に浮遊させ、シリンジを用いて培養バッグから培地を約0.1ml採取して、トリパンブルーで染色し血球計数盤を用いて細胞密度を計測した。なお、採取した細胞数は極少量であり、全体の細胞密度に影響はない。
また、インキュベータの使用と培地交換については、試験1と同様に行った。 However, in Test 3, unlikeTest 2, in order to measure the number of cells, the automatic liquid feeding was stopped, the culture bag was stirred to uniformly suspend the cells in the medium, and the medium was removed from the culture bag using a syringe. Approximately 0.1 ml was collected, stained with tripan blue, and the cell density was measured using a hemocytometer. The number of collected cells is extremely small, and there is no effect on the overall cell density.
The use of the incubator and the exchange of the medium were carried out in the same manner as in Test 1.
また、インキュベータの使用と培地交換については、試験1と同様に行った。 However, in Test 3, unlike
The use of the incubator and the exchange of the medium were carried out in the same manner as in Test 1.
試験3の細胞培養における、培養バッグに固定された蛍光式酸素濃度センサによる酸素濃度の測定結果を表すグラフを図6及び図7に示す。図6のグラフは1回目、図7のグラフは2回目の細胞数の計測までの酸素濃度の測定結果を表している。
FIGS. 6 and 7 show graphs showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of Test 3. The graph of FIG. 6 shows the measurement result of the oxygen concentration up to the first measurement of the cell number, and the graph of FIG. 7 shows the measurement result of the oxygen concentration up to the second measurement of the cell number.
本試験では、インキュベータ10の酸素濃度を指定のタイミングで増加させた。
具体的には、図6のグラフにおいて、測定開始から4時間後のタイミングで、21%から30%に変更した。また、測定開始から23時間後のタイミングで、30%から40%に変更した。さらに、図7のグラフにおいて、測定開始から6時間後のタイミングで、40%から50%に変更した。 In this test, the oxygen concentration of theincubator 10 was increased at a specified timing.
Specifically, in the graph of FIG. 6, it was changed from 21% to 30% at thetiming 4 hours after the start of measurement. In addition, it was changed from 30% to 40% at the timing 23 hours after the start of measurement. Furthermore, in the graph of FIG. 7, it was changed from 40% to 50% at the timing 6 hours after the start of measurement.
具体的には、図6のグラフにおいて、測定開始から4時間後のタイミングで、21%から30%に変更した。また、測定開始から23時間後のタイミングで、30%から40%に変更した。さらに、図7のグラフにおいて、測定開始から6時間後のタイミングで、40%から50%に変更した。 In this test, the oxygen concentration of the
Specifically, in the graph of FIG. 6, it was changed from 21% to 30% at the
そして、図6のグラフにおいて、測定開始42時間後に培養バッグを撹拌して細胞を培地中に均一に浮遊させ、培養バッグから培地を約0.1ml採取して、トリパンブルーで染色し血球計数盤を用いて細胞密度を計測した。その結果、測定開始42時間後の細胞密度は、4.04e6個/cm2であった。
Then, in the graph of FIG. 6, 42 hours after the start of measurement, the culture bag was stirred to uniformly float the cells in the medium, about 0.1 ml of the medium was collected from the culture bag, and stained with tripan blue to obtain a hemocytometer. The cell density was measured using. As a result, the cell density 42 hours after the start of measurement was 4.04e6 cells / cm 2 .
そして、図7のグラフにおいて、測定開始22.5時間後に培養バッグを撹拌して細胞を培地中に均一に浮遊させ、培養バッグから培地を約0.1ml採取して、トリパンブルーで染色し血球計数盤を用いて細胞密度を計測した。その結果、測定開始22.5時間後の細胞密度は、5.2e6個/cm2であった。
Then, in the graph of FIG. 7, 22.5 hours after the start of measurement, the culture bag was stirred to uniformly float the cells in the medium, about 0.1 ml of the medium was collected from the culture bag, stained with tripan blue, and a hemocytometer was obtained. The cell density was measured using. As a result, the cell density 22.5 hours after the start of measurement was 5.2e6 cells / cm 2 .
また、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法による細胞の単位面積当たりの個数の計算値は、以下のように算出される。
まず、ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))は、1.4e-2である。また、細胞1個の単位時間当たりの酸素消費量(mg/(hr・個))は、1.00e-9であった。 Further, the calculated value of the number of cells per unit area by the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells is calculated as follows.
First, the oxygen permeability (mg / (cm 2 · hr · atm)) of the gas permeable member is 1.4e-2. The oxygen consumption per unit time (mg / (hr ·)) of one cell was 1.00e-9.
まず、ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))は、1.4e-2である。また、細胞1個の単位時間当たりの酸素消費量(mg/(hr・個))は、1.00e-9であった。 Further, the calculated value of the number of cells per unit area by the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells is calculated as follows.
First, the oxygen permeability (mg / (cm 2 · hr · atm)) of the gas permeable member is 1.4e-2. The oxygen consumption per unit time (mg / (hr ·)) of one cell was 1.00e-9.
また、図6のグラフの測定開始42時間後において、培養容器の周囲の酸素濃度(%)は40%、酸素濃度センサの測定値(%)は、7%であった。
したがって、細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(40/100-7/100)/1.00e-9=4.62e6
これに対して、細胞密度の実測値は、上述のとおり、4.04e6個/cm2であった。 In addition, 42 hours after the start of measurement in the graph of FIG. 6, the oxygen concentration (%) around the culture vessel was 40%, and the measured value (%) of the oxygen concentration sensor was 7%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells is as follows.
C = 1.4e-2 × (40 / 100-7 / 100) /1.00e-9=4.62e6
On the other hand, the measured value of the cell density was 4.04e6 cells / cm 2 as described above.
したがって、細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(40/100-7/100)/1.00e-9=4.62e6
これに対して、細胞密度の実測値は、上述のとおり、4.04e6個/cm2であった。 In addition, 42 hours after the start of measurement in the graph of FIG. 6, the oxygen concentration (%) around the culture vessel was 40%, and the measured value (%) of the oxygen concentration sensor was 7%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells is as follows.
C = 1.4e-2 × (40 / 100-7 / 100) /1.00e-9=4.62e6
On the other hand, the measured value of the cell density was 4.04e6 cells / cm 2 as described above.
また、図7のグラフの測定開始22.5時間後において、培養容器の周囲の酸素濃度(%)は50%、酸素濃度センサの測定値(%)は、12%であった。
したがって、細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(50/100-12/100)/1.00e-9=5.32e6
これに対して、細胞密度の実測値は、上述のとおり、5.2e6個/cm2であった。 In addition, 22.5 hours after the start of measurement in the graph of FIG. 7, the oxygen concentration (%) around the culture vessel was 50%, and the measured value (%) of the oxygen concentration sensor was 12%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells is as follows.
C = 1.4e-2 × (50 / 100-12 / 100) /1.00e-9=5.32e6
On the other hand, the measured value of the cell density was 5.2e6 cells / cm 2 as described above.
したがって、細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=1.4e-2×(50/100-12/100)/1.00e-9=5.32e6
これに対して、細胞密度の実測値は、上述のとおり、5.2e6個/cm2であった。 In addition, 22.5 hours after the start of measurement in the graph of FIG. 7, the oxygen concentration (%) around the culture vessel was 50%, and the measured value (%) of the oxygen concentration sensor was 12%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells is as follows.
C = 1.4e-2 × (50 / 100-12 / 100) /1.00e-9=5.32e6
On the other hand, the measured value of the cell density was 5.2e6 cells / cm 2 as described above.
すなわち、本試験によっても、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法によれば、ガス透過性部材からなる培養容器によって細胞を静置状態で培養する場合において、細胞の単位面積当たりの個数の概算を算出できることが分かった。
That is, even in this test, according to the cell culture system of the present embodiment and the method for detecting cell proliferation, when cells are cultured in a stationary state in a culture vessel made of a gas permeable member, the cell unit. It was found that an approximate number of cells per area could be calculated.
[試験4]
培養バッグとして、厚み110μmの直鎖状低密度ポリエチレンからなり、外形のサイズが120mm×65mm、培養空間の底面積が48cm2である培養バッグ(東洋製罐グループホールディングス株式会社製)を準備した。 [Test 4]
As a culture bag, a culture bag (manufactured by Toyo Seikan Group Holdings Co., Ltd.) made of linear low-density polyethylene having a thickness of 110 μm, having an outer size of 120 mm × 65 mm and a bottom area of the culture space of 48 cm 2 was prepared.
培養バッグとして、厚み110μmの直鎖状低密度ポリエチレンからなり、外形のサイズが120mm×65mm、培養空間の底面積が48cm2である培養バッグ(東洋製罐グループホールディングス株式会社製)を準備した。 [Test 4]
As a culture bag, a culture bag (manufactured by Toyo Seikan Group Holdings Co., Ltd.) made of linear low-density polyethylene having a thickness of 110 μm, having an outer size of 120 mm × 65 mm and a bottom area of the culture space of 48 cm 2 was prepared.
この培養バッグは、図8に示すように、内側に複数の突起を備えると共に、外面に複数の小突起部が形成されたものである。具体的には、ピッチが0.11mm、高さが0.19mm、角度が75°の複数の略三角柱を山脈状に並列して間隔を空けずに加工した2枚のフィルムの周縁部をヒートシールすることによって形成した。フィルムの最も薄い部分(薄肉部)の厚みは、25μmである。この培養バッグにおける酸素透過度は、8.0e-2(mg/(cm2・hr・atm))であった。なお、この培養バッグの酸素透過度は、試験1で用いた培養バッグを例として上述した方法と同様にして算出した。
As shown in FIG. 8, this culture bag is provided with a plurality of protrusions on the inside and a plurality of small protrusions are formed on the outer surface. Specifically, the peripheral edges of two films processed in parallel in a mountain range with a pitch of 0.11 mm, a height of 0.19 mm, and an angle of 75 ° are heat-sealed. Formed by The thickness of the thinnest part (thin wall part) of the film is 25 μm. The oxygen permeability in this culture bag was 8.0e-2 (mg / (cm 2 · hr · atm)). The oxygen permeability of this culture bag was calculated in the same manner as described above using the culture bag used in Test 1 as an example.
この培養バッグ内面のほぼ中央に蛍光式酸素濃度センサを固定した。
また、本試験では押圧治具を用いることなく培養を行った。培養バッグの下側の蛍光式酸素濃度センサに対応する位置に蛍光受発光部を配置した。 A fluorescent oxygen concentration sensor was fixed in the center of the inner surface of the culture bag.
Moreover, in this test, the culture was carried out without using a pressing jig. A fluorescence receiving / emitting unit was placed at a position corresponding to the fluorescent oxygen concentration sensor on the lower side of the culture bag.
また、本試験では押圧治具を用いることなく培養を行った。培養バッグの下側の蛍光式酸素濃度センサに対応する位置に蛍光受発光部を配置した。 A fluorescent oxygen concentration sensor was fixed in the center of the inner surface of the culture bag.
Moreover, in this test, the culture was carried out without using a pressing jig. A fluorescence receiving / emitting unit was placed at a position corresponding to the fluorescent oxygen concentration sensor on the lower side of the culture bag.
細胞としては、iPS細胞(1231A3株,京都大学iPS細胞研究所)を1.7e4個/cm2の細胞密度で播種した。本試験では、培養バッグ内の上面と下面の両方を培養面として使用しており、この播種細胞密度は両面分である。
培地としては、StemFit(R)(AK-02N ,味の素ヘルシーサプライ株式会社)を使用し、培養バッグ内に35mlの培地を細胞と共に充填した。 As cells, iPS cells (1231A3 strain, Center for iPS Cell Research and Application, Kyoto University) were seeded at a cell density of 1.7e4 cells / cm 2 . In this test, both the upper surface and the lower surface in the culture bag are used as the culture surface, and the seeded cell density is for both sides.
As a medium, StemFit (R) (AK-02N, Ajinomoto Healthy Supply Co., Ltd.) was used, and 35 ml of the medium was filled in the culture bag together with the cells.
培地としては、StemFit(R)(AK-02N ,味の素ヘルシーサプライ株式会社)を使用し、培養バッグ内に35mlの培地を細胞と共に充填した。 As cells, iPS cells (1231A3 strain, Center for iPS Cell Research and Application, Kyoto University) were seeded at a cell density of 1.7e4 cells / cm 2 . In this test, both the upper surface and the lower surface in the culture bag are used as the culture surface, and the seeded cell density is for both sides.
As a medium, StemFit (R) (AK-02N, Ajinomoto Healthy Supply Co., Ltd.) was used, and 35 ml of the medium was filled in the culture bag together with the cells.
インキュベータとしては、高酸素培養可能なマルチガスインキュベータ(PHC株式会社,型式MCO-5M-PJ)を使用した。なお、本試験では、自動送液機構を使用せず、手作業により培地交換を行った。
培地を充填した培地供給バッグを10℃の保冷庫に保管すると共に、培養バッグにチューブで接続した。また、廃液バッグを培養バッグにチューブで接続した。培地の送液は、チューブポンプの制御により行った。培地は、37℃のインキュベータ内を通過する間に加温されて、培養バッグに入る時点では約30℃になった。 As an incubator, a multi-gas incubator capable of high oxygen culture (PHC Corporation, model MCO-5M-PJ) was used. In this test, the medium was changed manually without using the automatic liquid feeding mechanism.
The medium supply bag filled with the medium was stored in a cold storage at 10 ° C. and connected to the culture bag with a tube. In addition, the waste liquid bag was connected to the culture bag with a tube. The medium was fed under the control of a tube pump. The medium was warmed while passing through the 37 ° C incubator to about 30 ° C by the time it entered the culture bag.
培地を充填した培地供給バッグを10℃の保冷庫に保管すると共に、培養バッグにチューブで接続した。また、廃液バッグを培養バッグにチューブで接続した。培地の送液は、チューブポンプの制御により行った。培地は、37℃のインキュベータ内を通過する間に加温されて、培養バッグに入る時点では約30℃になった。 As an incubator, a multi-gas incubator capable of high oxygen culture (PHC Corporation, model MCO-5M-PJ) was used. In this test, the medium was changed manually without using the automatic liquid feeding mechanism.
The medium supply bag filled with the medium was stored in a cold storage at 10 ° C. and connected to the culture bag with a tube. In addition, the waste liquid bag was connected to the culture bag with a tube. The medium was fed under the control of a tube pump. The medium was warmed while passing through the 37 ° C incubator to about 30 ° C by the time it entered the culture bag.
そして、指定の時間に自動で培地の全量交換を実施することで、培養によって劣化した培地を培地バッグから排出し、新しい培地を充填した。
具体的には、培養開始から所定のタイミングで、培地交換を実施した。このとき、培地排出量は、35ml中35mlであり、送液速度は2ml/分とした。培地交換によって新培地が追加されると、培養バッグ内の酸素濃度が一時的に上昇し、後述するグラフにおいてピークが表示された。
また、本試験では、インキュベータ10の酸素濃度を増加させず、高酸素での培養は行わなかった。 Then, by automatically exchanging the entire amount of the medium at a designated time, the medium deteriorated by the culture was discharged from the medium bag and filled with a new medium.
Specifically, the medium was exchanged at a predetermined timing from the start of the culture. At this time, the amount of medium discharged was 35 ml out of 35 ml, and the liquid feeding rate was 2 ml / min. When new medium was added by medium exchange, the oxygen concentration in the culture bag temporarily increased, and a peak was displayed in the graph described later.
Moreover, in this test, the oxygen concentration of theincubator 10 was not increased, and the culture in high oxygen was not performed.
具体的には、培養開始から所定のタイミングで、培地交換を実施した。このとき、培地排出量は、35ml中35mlであり、送液速度は2ml/分とした。培地交換によって新培地が追加されると、培養バッグ内の酸素濃度が一時的に上昇し、後述するグラフにおいてピークが表示された。
また、本試験では、インキュベータ10の酸素濃度を増加させず、高酸素での培養は行わなかった。 Then, by automatically exchanging the entire amount of the medium at a designated time, the medium deteriorated by the culture was discharged from the medium bag and filled with a new medium.
Specifically, the medium was exchanged at a predetermined timing from the start of the culture. At this time, the amount of medium discharged was 35 ml out of 35 ml, and the liquid feeding rate was 2 ml / min. When new medium was added by medium exchange, the oxygen concentration in the culture bag temporarily increased, and a peak was displayed in the graph described later.
Moreover, in this test, the oxygen concentration of the
そして、測定開始63時間後に培養バッグから培地を除去した後、細胞剥離液(サーモフィッシャーサイエンティフィック株式会社,TrypLE)を用いて培養バッグから細胞を剥離した。そして、培養バッグに培地を充填して剥離した細胞を培地に懸濁させ、シリンジを用いて培養バッグから培地を全量回収して、トリパンブルーで染色し血球計数盤を用いて細胞密度を計測した。その結果、細胞密度は、3.20e6個/cm2であった。
試験4の細胞培養における、培養バッグに固定された蛍光式酸素濃度センサによる酸素濃度の測定結果を表すグラフを図9に示す。 Then, 63 hours after the start of measurement, the medium was removed from the culture bag, and then the cells were detached from the culture bag using a cell stripping solution (Thermo Fisher Scientific Co., Ltd., TrypLE). Then, the culture bag was filled with the medium, the detached cells were suspended in the medium, the entire amount of the medium was collected from the culture bag using a syringe, stained with trypan blue, and the cell density was measured using a hemocytometer. .. As a result, the cell density was 3.20e6 cells / cm 2 .
FIG. 9 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture ofTest 4.
試験4の細胞培養における、培養バッグに固定された蛍光式酸素濃度センサによる酸素濃度の測定結果を表すグラフを図9に示す。 Then, 63 hours after the start of measurement, the medium was removed from the culture bag, and then the cells were detached from the culture bag using a cell stripping solution (Thermo Fisher Scientific Co., Ltd., TrypLE). Then, the culture bag was filled with the medium, the detached cells were suspended in the medium, the entire amount of the medium was collected from the culture bag using a syringe, stained with trypan blue, and the cell density was measured using a hemocytometer. .. As a result, the cell density was 3.20e6 cells / cm 2 .
FIG. 9 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of
また、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法による細胞の単位面積当たりの個数の計算値は、以下のように算出される。
まず、ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))は、8.0e-2であった。また、細胞1個の単位時間当たりの酸素消費量(mg/(hr・個))は、2.34e-9であった。 Further, the calculated value of the number of cells per unit area by the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells is calculated as follows.
First, the oxygen permeability (mg / (cm 2 · hr · atm)) of the gas permeable member was 8.0e-2. The oxygen consumption per unit time (mg / (hr ·)) of one cell was 2.34e-9.
まず、ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))は、8.0e-2であった。また、細胞1個の単位時間当たりの酸素消費量(mg/(hr・個))は、2.34e-9であった。 Further, the calculated value of the number of cells per unit area by the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells is calculated as follows.
First, the oxygen permeability (mg / (cm 2 · hr · atm)) of the gas permeable member was 8.0e-2. The oxygen consumption per unit time (mg / (hr ·)) of one cell was 2.34e-9.
また、培養容器の周囲の酸素濃度(%)は21%、酸素濃度センサの測定値(%)は、12%であった。
したがって、細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=8.0e-2×(21/100-12/100)/2.34e-9=3.08e6
これに対して、細胞密度の実測値は、上述のとおり、3.20e6個/cm2であった。 The oxygen concentration (%) around the culture vessel was 21%, and the measured value (%) of the oxygen concentration sensor was 12%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells is as follows.
C = 8.0e-2 × (21 / 100-12 / 100) /2.34e-9=3.08e6
On the other hand, the measured value of the cell density was 3.20e6 cells / cm 2 as described above.
したがって、細胞の単位面積当たりの個数C(個/cm2)は、以下のとおりである。
C=8.0e-2×(21/100-12/100)/2.34e-9=3.08e6
これに対して、細胞密度の実測値は、上述のとおり、3.20e6個/cm2であった。 The oxygen concentration (%) around the culture vessel was 21%, and the measured value (%) of the oxygen concentration sensor was 12%.
Therefore, the number C (pieces / cm 2 ) per unit area of cells is as follows.
C = 8.0e-2 × (21 / 100-12 / 100) /2.34e-9=3.08e6
On the other hand, the measured value of the cell density was 3.20e6 cells / cm 2 as described above.
すなわち、本試験によっても、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法によれば、ガス透過性部材からなる培養容器によって細胞を静置状態で培養する場合において、細胞の単位面積当たりの個数の概算を算出できることが分かった。
That is, even in this test, according to the cell culture system of the present embodiment and the method for detecting cell proliferation, when cells are cultured in a stationary state in a culture vessel made of a gas permeable member, the cell unit. It was found that an approximate number of cells per area could be calculated.
[試験5]
本試験は、試験3に連続して行ったものであり、試験3における酸素濃度の測定に続く3日間の約60hrにおいて培養バッグ内の酸素濃度の測定を行った。
試験5の細胞培養における、培養バッグに固定された蛍光式酸素濃度センサによる酸素濃度の測定結果を表すグラフを図10に示す。 [Test 5]
This test was carried out in succession to Test 3, and the oxygen concentration in the culture bag was measured for about 60 hours for 3 days following the measurement of the oxygen concentration in Test 3.
FIG. 10 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture ofTest 5.
本試験は、試験3に連続して行ったものであり、試験3における酸素濃度の測定に続く3日間の約60hrにおいて培養バッグ内の酸素濃度の測定を行った。
試験5の細胞培養における、培養バッグに固定された蛍光式酸素濃度センサによる酸素濃度の測定結果を表すグラフを図10に示す。 [Test 5]
This test was carried out in succession to Test 3, and the oxygen concentration in the culture bag was measured for about 60 hours for 3 days following the measurement of the oxygen concentration in Test 3.
FIG. 10 shows a graph showing the measurement results of the oxygen concentration by the fluorescent oxygen concentration sensor fixed to the culture bag in the cell culture of
測定開始56時間後に培養バッグを撹拌して細胞を培地中に均一に浮遊させ、培養バッグから培地を約0.1ml採取して、トリパンブルーで染色し血球計数盤を用いて細胞密度を計測した。その結果、測定開始56時間後の細胞密度は、1.04e7個/cm2であった。
56 hours after the start of the measurement, the culture bag was stirred to uniformly float the cells in the medium, about 0.1 ml of the medium was collected from the culture bag, stained with trypan blue, and the cell density was measured using a blood cell counter. As a result, the cell density 56 hours after the start of measurement was 1.04e7 cells / cm 2 .
本試験では、図10に示されるように、測定開始25時間以降、培養バッグ内の細胞密度が非常に高密度になっている。特に測定開始56時間後の時点では、培養バッグ内の酸素濃度が1%未満になっている。
In this test, as shown in FIG. 10, the cell density in the culture bag is extremely high after 25 hours from the start of measurement. In particular, 56 hours after the start of measurement, the oxygen concentration in the culture bag was less than 1%.
このような場合、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法では、以下の方法によって、細胞の単位面積当たりの個数を計算する。
まず、培養バッグ内の細胞密度が非常に高密度になる前の培養時間において、培養バッグに複数回新培地を追加した時の酸素濃度の上昇ピーク値の変化の傾きPTと、培養バッグの安定時の酸素濃度の変化の傾きQTを計算し、その比率(QT/PT)を算出する。 In such a case, in the cell culture system of the present embodiment and the method for detecting the proliferation of cells, the number of cells per unit area is calculated by the following method.
First, in the culture time before the cell density in the culture bag becomes very high, the gradient PT of the change in the peak value of the increase in oxygen concentration when the new medium is added to the culture bag multiple times, and the stability of the culture bag. The gradient QT of the change in oxygen concentration at time is calculated, and the ratio (QT / PT) is calculated.
まず、培養バッグ内の細胞密度が非常に高密度になる前の培養時間において、培養バッグに複数回新培地を追加した時の酸素濃度の上昇ピーク値の変化の傾きPTと、培養バッグの安定時の酸素濃度の変化の傾きQTを計算し、その比率(QT/PT)を算出する。 In such a case, in the cell culture system of the present embodiment and the method for detecting the proliferation of cells, the number of cells per unit area is calculated by the following method.
First, in the culture time before the cell density in the culture bag becomes very high, the gradient PT of the change in the peak value of the increase in oxygen concentration when the new medium is added to the culture bag multiple times, and the stability of the culture bag. The gradient QT of the change in oxygen concentration at time is calculated, and the ratio (QT / PT) is calculated.
具体的には、図10において、P1(5.5, 36.3)とP2(20.1, 34.1)の上昇ピーク値の変化の傾きPTは、-15%(=(34.1-36.3)/(20.1-5.5)×100)であり、Q1(7.7, 9.3)とQ2(22.9, 2.6)の上昇ピーク値の変化の傾きQTは、-44%(=(2.6-9.3)/(22.9-7.7))であり、その比率(QT/PT)は、2.9である。
Specifically, in FIG. 10, the slope PT of the change in the rising peak value of P1 (5.5, 36.3) and P2 (20.1, 34.1) is -15% (= (34.1-36.3) / (20.1-5.5) ×. 100), and the slope QT of the change in the rising peak value of Q1 (7.7, 9.3) and Q2 (22.9, 2.6) is -44% (= (2.6-9.3) / (22.9-7.7)). The ratio (QT / PT) is 2.9.
次に、測定開始56時間後Q4(56, 1未満)の直前のピークは、P4(55.1, 22.3)であり、酸素濃度が計測できる時点Q3(31.1, 2.3)の直前のピークは、P3(29.5, 32.7)である。
Q4における酸素濃度は、P3からP4の酸素濃度の変化量に対して、上記比率(QT/PT)にもとづきQ3から変化すると推定することができる。
したがって、推定する酸素濃度D’は、以下のように計算することができる。
D’=Q3の酸素濃度-(P3からP4の酸素濃度の変化量)×QT/PT Next, the peak immediately before Q4 (less than 56, 1) 56 hours after the start of measurement is P4 (55.1, 22.3), and the peak immediately before Q3 (31.1, 2.3) when the oxygen concentration can be measured is P3 (55.1, 22.3). 29.5, 32.7).
It can be estimated that the oxygen concentration in Q4 changes from Q3 based on the above ratio (QT / PT) with respect to the amount of change in the oxygen concentration from P3 to P4.
Therefore, the estimated oxygen concentration D'can be calculated as follows.
D'= Oxygen concentration of Q3- (change in oxygen concentration from P3 to P4) x QT / PT
Q4における酸素濃度は、P3からP4の酸素濃度の変化量に対して、上記比率(QT/PT)にもとづきQ3から変化すると推定することができる。
したがって、推定する酸素濃度D’は、以下のように計算することができる。
D’=Q3の酸素濃度-(P3からP4の酸素濃度の変化量)×QT/PT Next, the peak immediately before Q4 (less than 56, 1) 56 hours after the start of measurement is P4 (55.1, 22.3), and the peak immediately before Q3 (31.1, 2.3) when the oxygen concentration can be measured is P3 (55.1, 22.3). 29.5, 32.7).
It can be estimated that the oxygen concentration in Q4 changes from Q3 based on the above ratio (QT / PT) with respect to the amount of change in the oxygen concentration from P3 to P4.
Therefore, the estimated oxygen concentration D'can be calculated as follows.
D'= Oxygen concentration of Q3- (change in oxygen concentration from P3 to P4) x QT / PT
すなわち、培養バッグに複数回新培地を追加した時の酸素濃度の上昇ピーク値の変化の傾きと培養バッグの安定時の酸素濃度の変化の傾きとの比率にもとづいて得られる所望の時点の培養バッグ内の酸素濃度の推定値D’を、酸素濃度センサの測定値として用いることで、以下の式(2)にもとづき細胞の単位面積当たりの個数を計算することができる。
C=G×(M/100-D’/100)/S ・・・式(2) That is, the culture at a desired time point obtained based on the ratio of the slope of the change in the peak value of the increase in oxygen concentration when the new medium was added to the culture bag multiple times and the slope of the change in the oxygen concentration when the culture bag was stable. By using the estimated value D'of the oxygen concentration in the bag as the measured value of the oxygen concentration sensor, the number of cells per unit area can be calculated based on the following formula (2).
C = G × (M / 100-D'/ 100) / S ... Expression (2)
C=G×(M/100-D’/100)/S ・・・式(2) That is, the culture at a desired time point obtained based on the ratio of the slope of the change in the peak value of the increase in oxygen concentration when the new medium was added to the culture bag multiple times and the slope of the change in the oxygen concentration when the culture bag was stable. By using the estimated value D'of the oxygen concentration in the bag as the measured value of the oxygen concentration sensor, the number of cells per unit area can be calculated based on the following formula (2).
C = G × (M / 100-D'/ 100) / S ... Expression (2)
よって、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法によれば、細胞の単位面積当たりの個数は、以下のように計算される。
ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))は、1.4e-2であった。また、細胞1個の単位時間当たりの酸素消費量(mg/(hr・個))は、1.00e-9であった。 Therefore, according to the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells, the number of cells per unit area is calculated as follows.
The oxygen permeability (mg / (cm 2 · hr · atm)) of the gas permeable member was 1.4e-2. The oxygen consumption per unit time (mg / (hr ·)) of one cell was 1.00e-9.
ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))は、1.4e-2であった。また、細胞1個の単位時間当たりの酸素消費量(mg/(hr・個))は、1.00e-9であった。 Therefore, according to the cell culture system of the present embodiment and the method for detecting the proliferativeness of cells, the number of cells per unit area is calculated as follows.
The oxygen permeability (mg / (cm 2 · hr · atm)) of the gas permeable member was 1.4e-2. The oxygen consumption per unit time (mg / (hr ·)) of one cell was 1.00e-9.
D’=2.3-(22.3-32.7)×2.9
=-27.9
C=1.4e-2×(50/100+27.9/100)/1.00e-9=1.09e7個/cm2
これに対して、細胞密度の実測値は、上述のとおり、1.04e7個/cm2であった。 D'= 2.3- (22.3-32.7) x 2.9
= -27.9
C = 1.4e-2 x (50/100 + 27.9 / 100) /1.00e-9=1.09e7 pieces / cm 2
On the other hand, the measured value of the cell density was 1.04e7 cells / cm 2 as described above.
=-27.9
C=1.4e-2×(50/100+27.9/100)/1.00e-9=1.09e7個/cm2
これに対して、細胞密度の実測値は、上述のとおり、1.04e7個/cm2であった。 D'= 2.3- (22.3-32.7) x 2.9
= -27.9
C = 1.4e-2 x (50/100 + 27.9 / 100) /1.00e-9=1.09e7 pieces / cm 2
On the other hand, the measured value of the cell density was 1.04e7 cells / cm 2 as described above.
すなわち、本試験によっても、本実施形態の細胞培養システム、及び細胞の増殖性の検出方法によれば、ガス透過性部材からなる培養容器によって細胞を静置状態で培養する場合において、細胞の単位面積当たりの個数の概算を算出できることが分かった。
That is, even in this test, according to the cell culture system of the present embodiment and the method for detecting cell proliferation, when cells are cultured in a stationary state in a culture vessel made of a gas permeable member, the cell unit. It was found that an approximate number of cells per area could be calculated.
本発明は、以上の実施形態及び実施例に限定されるものではなく、本発明の範囲内において、種々の変更実施が可能であることは言うまでもない。例えば、培養容器としては、例示したものに限定されず、例えば1ポートのものや3ポートを有するもの、培養面に複数のウェルを有するものなど培養の目的に応じて適宜変更したものを使用することなどが可能である。
It goes without saying that the present invention is not limited to the above embodiments and examples, and various modifications can be made within the scope of the present invention. For example, the culture container is not limited to the one illustrated, and for example, a container having one port, a container having three ports, a container having a plurality of wells on the culture surface, and the like, which are appropriately modified according to the purpose of the culture, are used. It is possible.
本発明は、細胞培養バッグを用いて細胞を高密度で大量培養する場合などに好適に利用することが可能である。
The present invention can be suitably used when cells are mass-cultured at high density using a cell culture bag.
この明細書に記載の文献及び本願のパリ優先の基礎となる日本出願明細書の内容を全てここに援用する。
The documents described in this specification and the contents of the Japanese application specification, which is the basis of the priority given to Paris in the present application, are all incorporated herein by reference.
10 インキュベータ
11 載置台
12 ガスセンサ
20,20a 培養容器
201,201a 上面ガス透過性フィルム
202,202a 下面ガス透過性フィルム
21,21a 蛍光式酸素濃度センサ
22,22a 蛍光受発光部
23a 穴あき樹脂部材
30 保冷庫
40 培地供給容器
50 廃液容器
60 制御装置
61 入出力部
62 蛍光式センサ入出力部
63 制御部
631 増殖検出部
64 操作部
65 電源部
H 溶着領域
i 細胞
L 培地
O 酸素ボンベ
C 二酸化炭素ボンベ 10 Incubator 11Stand 12 Gas sensor 20,20a Culture vessel 201,201a Top gas permeable film 202, 202a Bottom gas permeable film 21,21a Fluorescent oxygen concentration sensor 22, 22a Fluorescent light receiving / emitting unit 23a Perforated resin member 30 Cold insulation Storage 40 Medium supply container 50 Waste liquid container 60 Control device 61 Input / output unit 62 Fluorescent sensor input / output unit 63 Control unit 631 Proliferation detection unit 64 Operation unit 65 Power supply unit H Welding area i Cell L Medium O Oxygen bomb C Carbon dioxide bomb
11 載置台
12 ガスセンサ
20,20a 培養容器
201,201a 上面ガス透過性フィルム
202,202a 下面ガス透過性フィルム
21,21a 蛍光式酸素濃度センサ
22,22a 蛍光受発光部
23a 穴あき樹脂部材
30 保冷庫
40 培地供給容器
50 廃液容器
60 制御装置
61 入出力部
62 蛍光式センサ入出力部
63 制御部
631 増殖検出部
64 操作部
65 電源部
H 溶着領域
i 細胞
L 培地
O 酸素ボンベ
C 二酸化炭素ボンベ 10 Incubator 11
Claims (8)
- 少なくとも一部がガス透過性部材からなる培養容器によって細胞を静置状態で培養する細胞培養システムであって、
前記培養容器内における培養面付近の酸素濃度を計測する酸素濃度センサと、
前記ガス透過性部材の酸素透過度、前記酸素濃度センサの測定値、前記培養容器の周囲の酸素濃度、及び前記細胞の1個当たりの酸素消費量にもとづき前記細胞の増殖性を検出する増殖検出部と、を有する
ことを特徴とする細胞培養システム。 A cell culture system in which cells are cultured in a static state using a culture vessel made of at least a part of a gas permeable member.
An oxygen concentration sensor that measures the oxygen concentration near the culture surface in the culture vessel, and
Proliferation detection that detects the proliferation of the cells based on the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption per cell. A cell culture system characterized by having a portion and. - 前記増殖検出部が、前記細胞の増殖性として、前記細胞の単位面積当たりの個数を、以下の式(1)によって算出することを特徴とする請求項1記載の細胞培養システム。
C=G×(M/100-D/100)/S ・・・式(1)
C:細胞の単位面積当たりの個数(個/cm2)
G:ガス透過性部材の酸素透過度(mg/(cm2・hr・atm))
M:培養容器の周囲の酸素濃度(%)
D:酸素濃度センサの測定値(%)
S:細胞1個の単位時間当たりの酸素消費量(mg/(hr・個) ) The cell culture system according to claim 1, wherein the growth detection unit calculates the number of cells per unit area as the proliferative property of the cells by the following formula (1).
C = G × (M / 100-D / 100) / S ... Expression (1)
C: Number of cells per unit area (pieces / cm 2 )
G: Oxygen permeability of gas permeable member (mg / (cm 2・ hr ・ atm))
M: Oxygen concentration around the culture vessel (%)
D: Measured value (%) of oxygen concentration sensor
S: Oxygen consumption per unit time per cell (mg / (hr ·)) - 前記増殖検出部が、前記培養容器に複数回新培地を追加した時の酸素濃度の上昇ピーク値の変化の傾きと前記培養容器の安定時の酸素濃度の変化の傾きとの比率にもとづいて得られる所望の時点の前記培養容器内の酸素濃度の推定値を、前記酸素濃度センサの測定値として用いることを特徴とする請求項1又は2記載の細胞培養システム。 Obtained by the growth detection unit based on the ratio of the gradient of the change in the peak value of the increase in oxygen concentration when the new medium is added to the culture vessel a plurality of times and the gradient of the change in the oxygen concentration when the culture vessel is stable. The cell culture system according to claim 1 or 2, wherein an estimated value of the oxygen concentration in the culture vessel at a desired time point is used as a measured value of the oxygen concentration sensor.
- 前記培養容器の周囲の酸素濃度が、前記培養容器が収容されたインキュベータ内の酸素濃度、又は、大気中の酸素濃度である
ことを特徴とする請求項1~3のいずれかに記載の細胞培養システム。 The cell culture according to any one of claims 1 to 3, wherein the oxygen concentration around the culture vessel is the oxygen concentration in the incubator containing the culture vessel or the oxygen concentration in the atmosphere. system. - 前記酸素濃度センサが、蛍光式であることを特徴とする請求項1~4のいずれかに記載の細胞培養システム。 The cell culture system according to any one of claims 1 to 4, wherein the oxygen concentration sensor is a fluorescent type.
- 前記酸素濃度センサが、前記培養容器内における培養面に穴あき樹脂部材を用いて固定されていることを特徴とする請求項1~5のいずれかに記載の細胞培養システム。 The cell culture system according to any one of claims 1 to 5, wherein the oxygen concentration sensor is fixed to the culture surface in the culture container by using a perforated resin member.
- 前記培養容器及び前記穴あき樹脂部材が、ポリエチレン、ポリプロピレン、エチレンとα-オレフィンの共重合体、エチレンと酢酸ビニルの共重合体、エチレンとアクリル酸やメタクリル酸共重合体と金属イオンを用いたアイオノマーから選択されるいずれかからなることを特徴とする請求項1~6のいずれかに記載の細胞培養システム。 The culture vessel and the perforated resin member used polyethylene, polypropylene, a copolymer of ethylene and α-olefin, a copolymer of ethylene and vinyl acetate, and a copolymer of ethylene and acrylic acid or a methacrylic acid copolymer and metal ions. The cell culture system according to any one of claims 1 to 6, wherein the cell culture system comprises any of the ionomers.
- 少なくとも一部がガス透過性部材からなる培養容器によって細胞を静置状態で培養する細胞培養における細胞の増殖性の検出方法であって、
前記培養容器内における培養面に配置された酸素濃度センサにより前記培養容器内における培養面付近の酸素濃度を計測し、
前記ガス透過性部材の酸素透過度、前記酸素濃度センサの測定値、前記培養容器の周囲の酸素濃度、及び前記細胞の1個当たりの酸素消費量にもとづき前記細胞の単位面積当たりの個数を計算する
ことを特徴とする細胞培養における細胞の増殖性の検出方法。
A method for detecting cell proliferation in cell culture in which cells are cultured in a stationary state by a culture vessel made of at least a part of a gas permeable member.
The oxygen concentration near the culture surface in the culture vessel is measured by the oxygen concentration sensor arranged on the culture surface in the culture vessel.
The number of cells per unit area is calculated based on the oxygen permeability of the gas permeable member, the measured value of the oxygen concentration sensor, the oxygen concentration around the culture vessel, and the oxygen consumption per cell. A method for detecting the proliferativeness of cells in a cell culture, which comprises the above.
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JPS6315150A (en) | 1986-07-08 | 1988-01-22 | Terumo Corp | Method and apparatus for measuring viable cell number |
JPH06121667A (en) | 1992-10-13 | 1994-05-06 | Shimadzu Corp | Cell incubator |
JP2015516175A (en) * | 2012-05-18 | 2015-06-11 | ウィルソン ウォルフ マニュファクチャリング コーポレイションWilson Wolf Manufacturing Corporation | Improved cell culture method for adoptive cell therapy |
US20190218492A1 (en) * | 2016-09-14 | 2019-07-18 | Yale University | Self regulating bioreactor apparatus and methods |
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JPS6315150A (en) | 1986-07-08 | 1988-01-22 | Terumo Corp | Method and apparatus for measuring viable cell number |
JPH06121667A (en) | 1992-10-13 | 1994-05-06 | Shimadzu Corp | Cell incubator |
JP2015516175A (en) * | 2012-05-18 | 2015-06-11 | ウィルソン ウォルフ マニュファクチャリング コーポレイションWilson Wolf Manufacturing Corporation | Improved cell culture method for adoptive cell therapy |
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